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DNA Analysis
Notebook
Table of Contents
Genomic DNA Purification
2
Overview
Wizard® Genomic DNA Purification Kit
Wizard® SV and SV 96 Genomic DNA
Purification Systems
MagneSil® Blood Genomic, Max Yield System
MagneSil® ONE, Fixed Yield Blood Genomic System
MagneSil® Genomic, Fixed Tissue System
MagneSil® KF, Genomic System
Wizard® Magnetic 96 DNA Plant System
Ordering Information
Amplifying DNA
13
Overview
Optimization of PCR
Routine PCR
Proofreading Polymerases
Hot Start Methodology
dNTPs
Ordering Information
www.promega.com • techserv@promega.com
PCR Clean-Up
28
Overview
Wizard® SV Gel and PCR Clean-Up System
Wizard® SV 96 PCR Clean-Up System
Wizard® MagneSil® PCR Clean-Up System
Wizard® PCR Preps DNA Purification System
Ordering Information
Cloning PCR DNA
34
Overview
Basic Subcloning
Direct Mammalian Expression
Direct Bacterial Expression
PCR Cloning Techniques
Ordering Information
DNA Analysis Tools
Benchtop DNA Markers
Ladders and Digest Markers
43
Genomic DNA Purification
Please note, in the flow charts that follow,
Promega systems are listed by which protocols
come in the Technical Literature sent with the
system. Other applications to other gDNA sources
may exist (e.g., Blood protocols for the
Wizard SV Genomic System).
Look for these symbols to find the system right
for your application.
Blood
Cultured Cells
Animal Tissue
Plant Tissue
Bacteria
Yeast
Fixed Tissue
Buffy
coat or
white
blood c
ells?
Use cu
lt
cells p ured
rotocol
s.
Blood
Please contact Promega Technical Services
if you have questions.
techserv@promega.com
0.05–0.2ml
Requires automation,
magnetics-based
system. Specifically
designed to capture
just ~1µg gDNA
from 50–60µl of
blood. Eliminates
need to quantitate
after purification.
Requires
automati
magneti
cs-based on,
system.
Will cap
ture >
_11µg of
gDNA f
rom a 2
00µl
blood sa
mple.
2
0.2–10ml
Automated
MagneSil® ONE,
Fixed Yield Blood
Genomic System
MagneSil® Blood
Genomic, Max
Yield System
NEW
Manual
MagneSil®
KF, Genomic
System
Wizard®
Genomic DNA
Purification Kit
Designed for automation on
the KingFisher® mL.
Handles up to 200µl of
blood. Will purify 4-6µg of
gDNA from 1–15 samples
in 25 minutes.
3981MA02_3A
Promega has a variety of solutions for your genomic
DNA (gDNA) purification needs. Promega has
automated methods for blood, cultured cells,
mammalian tissue and plant purifications. All systems
produce high-quality DNA ready for amplification. Most
automated systems use magnetic particle-based
technologies. The Wizard® SV technologies introduce
membrane-based purification for manual or automated
purifications. The Wizard Genomic DNA Purification Kit
is a truly versatile solution-based system to manually
isolate high molecular weight gDNA from a variety of
starting materials. This gentle solution-based method
produces gDNA suitable for amplification, Southern
blotting and genomic cloning.
lly
ased, tota
b
n
io
t
lu
o
S
es
ystem. Us
scalable s Can handle
tion.
centrifuga blood. Specific
of
any volume provided for
protocols
ml of
ml and 10
300µl, 3 ole blood.
fresh wh
Promega DNA Analysis Notebook
Genomic DNA Purification
Automated
Manual
Wizard® SV 96
Genomic DNA
Purification System
Wizard® SV
Genomic DNA
Purification System
Wizard® Genomic
DNA Purification Kit
y
otall
t
,
d
base
ses
n
o
U
i
t
.
u
l
m
So
s
syste
duce
e
o
l
r
b
a
P
l
sca
tion.
t
a
g
u
f
i
eigh
r
w
t
n
r
ce
e
ula
itabl
olec
u
s
m
high
DNA
g
)
kb
tion,
a
c
(>50
i
l
pp
ny a
nd
a
r
R a
fo
C
P
.
ding
u
l
c
tting
n
i
o
l
b
hern
t
u
o
S
3982MA02_3A
Cultured
Cells
h a
Use wit uge or
ntrif
microce nifold. Purify
ma
vacuum ady gDNA in
PCR-re s after lysis.
te
20 minu rocess up to
Can p 6 cells/prep.
5 x 10
Perform 96 gDNA preps
at once. Requires a vacuum
manifold. Can be used on
benchtop or automated.
Comprised of SV
membranes arrayed in a
96-well format. Can
handle up to 5 x
Yeast
106
Bacteria
cells/well.
Manual
Genome Size
2.9Gb
2.7Gb
2.8Gb
3.0Gb
4.4Gb
2.5Gb
16.0Gb
13.5Mb
4.7Mb
Molecules/µg
3.13 × 105
3.35 × 105
3.26 × 105
3.04 × 105
2.07 × 105
3.65 × 105
5.70 × 104
6.75 × 107
1.94 × 108
Gb = Gigabases (1 × 109). Mb = Megabases (1 × 106).
www.promega.com • techserv@promega.com
You supply
lyticase and
50mM EDTA
Grampositive
Gramnegative
You supply
lysozyme and/or
lysostaphin
3985MA02_3A
Human
Mouse
Rat
Arabidopsis
Tobacco
Corn
Wheat
Yeast (S. cerevisiae)
Bacteria (E. coli)
Manual
Wizard® Genomic
DNA Purification Kit
3
Genomic DNA Purification
Plant
Tissue
Manual
Requires a 96-well
grinding apparatus
Requires liquid
nitrogen, mortar & pestle
Wizard® Magnetic
96 DNA
Plant System
Wizard® Genomic
DNA Purification Kit
3984MA02_3A
Automated
Magnetic
-based sy
stem that
be used i
can
n manual o
r
a
utomated
format. Pu
rifies gDN
A from
seeds, lea
f or tissu
es for PC
or other
R
amplificatio
n
based
genotyping
methods.
4
ally
sed, tot
a
b
n
io
t
Solu
or fresh
f
m
e
t
s
sy
scalable
ses
issue. U
t
f
a
le
plant
es
. Produc
n
io
t
a
g
u
centrif
t
r weigh
la
u
c
le
o
high m
suitable
A
N
D
g
)
(>50kb
n,
pplicatio
a
y
n
a
r
fo
d
PCR an
g
n
i
d
lu
inc
blotting .
n
r
e
h
t
Sou
Don’t see your DNA sample
type in these flowcharts?
Contact Promega Technical
Services for advice at:
techserv@promega.com
Promega DNA Analysis Notebook
Genomic DNA Purification
ne
No Xyle
for
required
ation!
z
i
n
i
f
f
a
r
depa
Manual
Overnight
proteinase K
digestion
Overnight
proteinase K
digestion
(you supply the enzyme)
(you supply the enzyme)
Wizard® SV 96
Genomic DNA
Purification System
Wizard® SV
Genomic DNA
Purification System
Wizard® Genomic
DNA Purification Kit
calable
s
y
l
l
a
t
o
based, t
n
o
ation.
i
t
g
u
u
l
f
o
i
r
S
t
n
Uses ce
.
m
weight
e
t
s
r
y
a
l
s
u
c
e
l
high mo
s
or any
e
f
c
u
e
l
d
b
a
t
Pro
i
u
gDNA s
and
)
b
R
k
C
0
P
g
(>5
n
, includi
n
o
i
t
a
c
i
l
app
lotting .
b
n
r
e
h
Sout
Use with a
microcentrifuge or vacuum
manifold with vacuum
adapters. Get PCR-ready
gDNA in 20 minutes
after lysis. Can process up
to 20mg tissue/prep.
Fixed
Tissue
Manual
Overnight
proteinase K
MagneSil®
Genomic, Fixed
Tissue System
www.promega.com • techserv@promega.com
3983MA02_3B
Perform
s 96 g
DNA
preps a
t once
. Uses
vacuum
a
manifol
d
.
Can
be use
bencht d on the
op or a
utomate
Compri
d.
sed of
membr
S
V
anes ar
r
a
yed in
a 96-w
e
l
l
f
ormat.
Proces
ses up
to
tissue/w 20mg
ell.
Automated
3983MA02_3A
Animal
Tissue
orks
w
m
yste ions.
s
d
base in sect ified
s
c
neti 0µm th ion-qual as
g
a
1
M
at
rs
with amplific amplime in
ies
ks
ng
Purif allowi b. Wor ns.
k
A
o
gDN e as 1.8 plificati
larg iplex am
mult
5
Genomic DNA Purification
Wizard® Genomic DNA
Purification Kit
Wizard® Genomic DNA Purification Kit
Need a versatile genomic DNA purification system?
Get it all with the Wizard Genomic DNA Purification Kit.
This solution-based system uses a simple, gentle
“salting out” method to isolate genomic DNA from a
wide variety of starting materials. The standard protocol
allows isolation of gDNA from blood or cultured cells.
Simple modifications to the protocol (involving the
addition of reagents common to most molecular
biology laboratories) allow you to isolate gDNA from
bacteria, yeast, plant or animal tissues, including
mouse tails. The system isolates high molecular weight
DNA (>50kb) with an A260/A280 greater than 1.7.
Protocols provided for:
Whole Blood
(300µl, 3ml, 10ml, and 50µl × 96-wells)
Cultured Cells
Animal Tissue
Plant Tissue
Gram-Negative Bacteria
Gram-Positive Bacteria
Cat.#: A1120 (100 isolations, 300µl blood)
A1125 (500 isolations, 300µl blood)
A1620 (100 isolations, 10ml blood)
Protocol:
www.promega.com/tbs/tm050/tm050.html
Customizable Protocol:
www.promega.com/tbscustom/tm050c/promega.asp
Citations detailing use of this kit:
www.promega.com/citations/
DNA Yields from Various Starting Materials.
Amount of
Source
Starting Material
Whole Blood
300µl
3ml
10ml
96-well plate, 50µl/well
Tissue Culture Cells
106–107 cells
Animal Tissue
Mouse Liver
11mg
Mouse Tail
0.5–1cm of tail
Insect Cells
5 × 106 cells
Plant Leaf Tissue
40mg
Bacterial Culture*
108–1010 cells
Yeast*
1.9 × 108 cells
Typical
DNA Yield
5–15µg
25–50µg
250–500µg
0.2–0.7µg
5–30µg
15–20µg
10–30µg
16µg
7–12µg
5–20µg
4.5–6.5µg
*Overnight culture.
Yeast
ut
nc
u
A,
N
aD
d
mb
Ra
t
cu
un
D
ell
PC
c
12
,
NA
t
cu
un
NA
D
aD
d
mb
La
1010MA04_5B
La
in
ra
tb
,
NA
The Wizard Genomic DNA Purification Kit has been
cited for purification of gDNA from the following
bacterial sources:
Bordetella, Borrelia, Campylobacter, Desulfovibrio,
Escherichia, Flavobacterium, Haemophilus,
Helicobacter, Leptospira, Methanococcus,
Mycobacterium, Mycoplasma, Paracoccus,
Prevotella, Proteus, Rickettsia, Salmonella,
Serratia, Sphingomonas, Staphylococcus,
Streptococcus, Treponema and Vibrio.
Citations describing isolation from these and other
sources, including yeast, fungi and virus-infected
cells, are available online at:
www.promega.com/citations/
Genomic DNA isolated using the Wizard Genomic DNA Purification Kit.
Genomic DNA was isolated from fresh rat brain or PC12 cells according to the protocol
provided in the Wizard ® Genomic DNA Purification Kit Technical Manual #TM050. DNA
from the indicated sources (0.5µg/lane) was separated on a 0.7% agarose gel.
6
c
enomi
G
d
r
Wiza ication Kit he
e
h
T
Purif
for t
DNA protocols , manual
es
ed
provid solution-bas NA from
D
easy, ation of g sources.
c
purifi different
many
Promega DNA Analysis Notebook
The Wizard SV and SV 96 Genomic DNA Purification
Systems provide a fast, simple technique for the
preparation of genomic DNA from cultured cells and
tissue, including mouse tails. The SV system is
designed for single-prep manual applications using
either a microcentrifuge or vacuum manifold. Genomic
DNA is obtained in 20 minutes after cell or tissue lysis.
The SV 96 system was developed to meet highthroughput needs. You can use this system on the
benchtop for manual 96-well purifications or automate
on a liquid-handling platform like the Beckman Coulter
Biomek® FX or Biomek® 2000 with a suitable vacuum
manifold. Both systems provide similar yields of high
quality, PCR-ready genomic DNA. Isolation of DNA from
tissue requires the additional purchase of DNase-free
Proteinase K (e.g., Promega Cat.# V3021).
M
–C +C
3710TA04_2A
Wizard® SV and SV 96
Genomic DNA Purification
Systems
Sp
in
Sp Tai
in l
L
Sp ive
in r
Sp Hea
in rt
Va Bra
c u in
Va um
cu Ta
Va um il
c u L iv
Va um er
c u He
um ar
t
Br
ai
n
Genomic DNA Purification
Amplification of genomic DNA isolated from various mouse tissue sources
using the Wizard SV Genomic DNA Purification System. Genomic DNA was
isolated from the tissues listed using either the vacuum or spin protocols provided in the
Wizard SV Genomic DNA Purification System Technical Bulletin #TB302. One microliter of
the eluate from the column was amplified for a mouse IL-1β (1.2kb) product. The positive
control (+C) was Mouse Genomic DNA (Cat.# G3091) and the negative control (–C)
contained no DNA. Further details are provided in Grunst, T. and Worzella, T. (2002)
Introducing the Wizard SV and SV 96 Genomic DNA Purification Systems. Promega Notes
81, 9–13.
Genomic DNA
Purification from
Mouse Tail Clipping
or Tissue Sample
Wizard® SV Genomic DNA Purification System
Mouse tail clipping
or tissue sample
Proteinase K
(Cat.# V3021)*
digestion in
Digestion Solution.
Cat.#: A2360 (50 preps)
A2361 (250 preps)
Incubate at 55°C
overnight
(16–18 hours).
Add Wizard®
SV Lysis Buffer.
Protocol:
www.promega.com/tbs/tb302/tb302.html
Genomic DNA
Purification from
Tissue Culture Cells
Wash tissue
culture cells
with 1X PBS.
Add Wizard®
SV Lysis Buffer.
Transfer lystate to minicolumn.
Want to use the SV Genomic Systems for manual
gDNA purification from blood?
Centrifuge.
Vacuum Adapter
(Cat.# A1331)*
Request Genomic DNA Purification from Blood:
Wizard ® SV Genomic DNA Purification Systems.
Application Note #AN101:
Vac-Man®
Laboratory
Vacuum Manifold
(Cat.# A7231)*
Bind DNA.
Wash, removing
solution by
centrifugation
or vacuum.
Transfer spin
column to a 1.5ml
microcentrifuge
tube (not provided).
Centrifuge.
3643MA02_2A
Elute genomic DNA.
Overview of the Wizard SV Genomic DNA Purification System spin and
vacuum protocols. *Vacuum Adaptor, Vacuum Manifold and Proteinase K must be
purchased separately.
www.promega.com • techserv@promega.com
7
Genomic DNA Purification
Average Yield of Genomic DNA Purified from Various
Sources Using the Wizard® SV and SV 96 Genomic DNA
Purification Systems.
Sample Type
Mouse Tail Clipping
Mouse Liver
Mouse Heart
Mouse Brain
CHO Cells
NIH3T3 Cells
293 Cells
Starting Amount
20mg
20mg
20mg
20mg
1 × 106 cells
1 × 106 cells
1 × 106 cells
Work wit
h the ter
minal 2cm
mouse tail
of
s. Any hig
her up on
and you'll
the tail
get more
c
o
n
n
ective tis
cartilage
sue,
and bone
t
h
a
n
n
u
cleated c
This mate
ells.
rial not on
ly
c
lo
g
s columns,
but fewer
cells mea
ns less gD
NA.
Average Yield
20µg
15µg
10µg
6µg
5µg
9µg
8µg
Average Yield (µg)
35
30
1
2
3
4
5
6
7
8
9
10 11 12
A
25
B
C
20
D
15
E
F
10
G
H
5
Mouse Tail Clippings
0
SV 96
SV Vacuum
SV Spin
Method of Purification
Water Only
M –C +C C4 C5 C6 C7 C8 D4 D5 D6 D7 D8
M
E4 E5 E6 E7 E8
Wizard SV 96 Genomic DNA
Purification System
3712TA04_2A
Comparison of DNA yields using the Wizard SV and SV 96 Genomic DNA
Purification Systems. Average yield of genomic DNA (µg) purified from 20mg mouse
tail clippings (1.2cm tail tip portions). Average A260/A280 ratios are: SV 96, 1.7 ± 0.08;
SV Vacuum, 1.7 ± 0.14; and SV Spin, 1.7 ± 0.14.
Cross-contamination assay. Genomic DNA was purified from mouse tail clippings or
water samples arrayed in adjacent wells of a 96-well plate using the Wizard SV 96 Genomic
DNA Purification System. PCR products were amplified from 1µl of purified sample from
each well for mouse IL-1β (1.2kb). No product is expected from wells containing water. For
further details, see Grunst, T. and Worzella, T. (2002) Introducing the Wizard SV and SV 96
Genomic DNA Purification Systems. Promega Notes 81, 9–13.
Cat.#: A2370 (1 × 96 preps)
A2371 (4 × 96 preps)
Protocol:
www.promega.com/tbs/tb303/tb303.html
The Wizard SV 96 Genomic System is suitable for
manual DNA purification at the benchtop or can be
easily automated on liquid handlers such as the
Biomek® FX, Biomek® 2000, or MultiPROBE® II HT/EX.
nto
i
s
n
r
tu
Robot nomic
a ge ication
purif
A
N
D
ine!
mach
8
3446CA06_1A
For more information visit:
www.promega.com/automethods/
Promega DNA Analysis Notebook
Genomic DNA Purification
MagneSil® Blood Genomic,
Max Yield & MagneSil ONE,
Fixed Yield Blood Genomic
4031CA03_3A
Promega has developed two genomic DNA isolation
systems to streamline your blood-to-analysis pathway—
MagneSil Blood Genomic, Max Yield(a) and MagneSil
ONE, Fixed Yield Blood Genomic(a). Both systems are
designed for automated gDNA purification on liquidhandling workstations such as the Beckman Courlter
Biomek® FX. The Max Yield System purifies ≥4µg of
gDNA from 200µl of whole blood. The MagneSil ONE
System purifies 1µg gDNA (±50%) from 60µl whole
blood. Both systems produce gDNA that is ready for use
in monoplex or multiplex PCR amplification reactions.
Fixed Yie
ld
quantitatio means no more
n or norm
alization!
Optimize
the volum
e
eluted D
NA y ou n of
e ed
o n ce — u s e
th
am o u n t e a a t s am e
ch time.
Analysis of DNA isolated using the MagneSil Blood Genomic, Max Yield System. DNA isolated from whole blood using the MagneSil Blood Genomic, Max Yield System was
used with the PowerPlex® 16 System (Cat.# DC6531), a multiplex STR amplification system for use in DNA typing. Results show successful coamplification and 3-color detection of the 16 loci
(15 STR loci plus amelogenin) in the PowerPlex® 16 System(b,c,d). Amplification products were separated on an ABI PRISM® 310 Genetic Analyzer, and analyzed using GeneScan® Software.
MagneSil® Blood Genomic, Max Yield System
MagneSil® ONE, Fixed Yield Blood Genomic System
Cat.#: MD1360 (1 × 96 preps)
Cat.#: MD1370 (1 × 96 preps)
Protocol:
www.promega.com/tbs/tb312/tb312.html
Protocol:
www.promega.com/tbs/tb313/tb313.html
For more information on automated methods visit:
www.promega.com/automethods/
For more information on automated methods visit:
www.promega.com/automethods/
ge of
a
r
o
t
s
m
r
Long-te
A
ute gDN
l
E
?
A
gDN
on like
i
t
u
l
o
s
r
e
in a buff
A
r or DN
e
f
f
u
b
TE
Solution
n
o
i
t
a
r
Rehyd
.
A7963)
.#
t
a
C
(
rage in
o
t
s
m
r
e
Long-t
lead to
n
a
c
r
wate
radation
g
e
d
c
i
autolyt
gDNA.
of the
Purify u
p to _> 4
µg
gDNA f
rom 20
0µl
whole b
lood.
www.promega.com • techserv@promega.com
gDNA
g
µ
1
~
Purify
60µl
–
0
5
from
lood.
whole b
DNA Yields (ng) From 60µl Whole Blood Samples Using
the MagneSil ONE, Fixed Yield Blood Genomic System.
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Mean
SD
Donor 1 Donor 2 Donor 3 Donor 4 Donor 5
1078
1078
1231
1451
1025
990
1092
1256
1078
998
970
1047
1315
990
994
1209
1294
1047
970
967
1105
1063
1388
1209
843
839
843
1296
1105
797
1032
1070
1256
1134
937
128
143
116
178
94
9
Genomic DNA Purification
MagneSil® Genomic,
Fixed Tissue System
Use your Thermo Electron KingFisher® mL to the fullest
with the MagneSil KF, Genomic System. With this
system, we provide optimized lysis and wash buffers to
allow purification of high quality genomic DNA in the
fixed number of steps required on the KingFisher®
instrument. The MagneSil KF, Genomic System purifies
2–6µg DNA from 200µl blood. The isolated DNA is ready
for monoplex and multiplex analysis, STR analysis and
SNP genotyping.
1 2
3 4
5
6 7
8 9 10 11 12 13 14 15 16 17
4212TA06_3A
Do you have archived samples of formalin-fixed,
paraffin-embedded tissues that you’d like to genotype?
We have developed a system that extracts amplifiable
gDNA from 10µm sections without xylene extraction.
The new MagneSil Genomic, Fixed Tissue System uses
proteinase K digestion followed by a rapid one hour
processing step to prepare samples for amplification.
The purification process removes amplification
inhibitors such as small DNAs, and isolates gDNA
fragments large enough to allow amplification targets up
to 1.8kb. The highly pure gDNA can be used in
monoplex or multiplex amplification reactions.
MagneSil® KF,
Genomic System
MagneSil® Genomic, Fixed Tissue System
Cat.#: MD1490 (100 samples)
Protocol:
www.promega.com/tbs/tb319/tb319.html
Yield gel of gDNA isolated from 200µl of liquid blood. A single run of 15 samples
prified on the KingFisher® mL using the MagneSil KF, Genomic System. The microliters of
each eluate were loaded on the gel and visualized by ethidium bromide staining. Lanes 6
and 12 intentionally left blank.
NEW
MagneSil® KF, Genomic System
A.
Cat.#: MD1460 (200 preps)
Protocol:
www.promega.com/tbs/tb322/tb322.html
C.
Thermo Electron KingFisher® mL tube strips
972 base
Amelogenin
Fragment
200µl
sample
200µl
MagneSil®,
KF PMPs
APC
1.8Kb
Fragment
1,000µl
Salt Wash,
Blood
1,000µl
Alcohol Wash,
Blood
1,000µl
Alcohol Wash,
Blood
200µl
NucleaseFree Water
800µl
Lysis Buffer,
KF
4106MA04_3A
Size Markers
926 base
Size
Marker
3931TC12_2A
B.
NEW
lity
a
u
q
iplex
t
l
u
M
from
A
gDN issue
t
fixed
10
Amplify targets
as large as 1.8Kb!
25 m
inutes
from
start
to fin
ish fo
r
1–15
sample
s
Promega DNA Analysis Notebook
Genomic DNA Purification
The Wizard Magnetic 96 DNA Plant System(a) is
designed for manual or automated 96-well purification
of genomic DNA from plant leaf and seed tissue. The
system was initially validated with corn and tomato leaf
as well as with canola and sunflower seeds. The DNA
purified from these samples can be used in PCR as well
as more demanding applications such as Rapid
Amplification of Polymorphic DNA (RAPD) analysis. The
Wizard Magnetic 96 DNA Plant System uses MagneSil®
Paramagnetic Particles(a) (PMPs), considered a “mobile
solid phase”. The binding of nucleic acids to magnetic
particles occurs in solution, resulting in increased
binding kinetics and binding efficiency. Contact with the
wash buffer is also enhanced, facilitating removal of
contaminants and increasing nucleic acid purity.
Automated methods for the Beckman Coulter Biomek®
2000, Biomek® FX and other robots are available.
Plant Sample Types Processed Using the Wizard
Magnetic 96 DNA Plant System.
Arapidopsis
Cotton seed
Soybean
Cabbage seed
Grass seed
Squash
Canola leaf
Green pepper seed
Squash seed
Canola seed
Lettuce
Strawberry leaf*
Carrot seed
Milkweed leaf
Sunflower seed
Chicory leaf
Potato tuber
Tobacco seedling
Chives
Radish leaf
Tomato leaf
Corn leaf
Rice leaf
Tomato seed
Cotton leaf*
Sorghum
Watermelon seed
*These samples require addition of polyvinylpolypyrrolidone (PVPP) to the
lysis buffer to remove phenolic compounds that inhibit PCR.
Typical DNA Yield from Plant Species Using the Wizard
Magnetic 96 DNA Plant System.
Arabidopsis tissue
10ng/mg
Canola leaf punches* (12)
26ng/leaf punch
Canola seeds (5)
343ng/seed
Corn leaf punches* (12)
98ng/leaf punch
Cotton seed (1)
29ng/seed
Lettuce leaf punches* (8)
13ng/leaf punch
Melon seed (1)
166ng/seed
Radish leaf punches* (12)
89ng/leaf punch
Soybean (10mg)
10ng/mg bean
Squash seed (1)
279ng/seed
Sunflower seed (1)
405ng/seed
Tomato leaf punches* (12)
111ng/leaf punch
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
bp
1,500 –
500 –
3315TB03_1A
Wizard® Magnetic 96 DNA
Plant System
100 –
1
2
3
4
5
6
7
8
Tobacco seedling
Soybean
Lettuce leaf
Corn leaf
Potato
Arabidopsis leaf
Cabbage seed
Green pepper seed
9
10
11
12
13
14
15
Prairie grass, seed head
Chives
Tomato leaf
Rice
Canola seed
Sunflower seed
Carrot seed
The Wizard Magnetic 96 DNA Plant System produces PCR-quality DNA from a
variety of plant species. One microliter of gDNA purified from the indicated materials
was used as template in PCR using a universal primer pair specific for the intron of the
TrnL chloroplast gene. One or more bands are produced depending on the plant species.
For more information, please see Koller, S. et al. (2001) Automated genomic DNA
purification using the Wizard Magnetic 96 DNA Plant System. Promega Notes 79, 25–28.
Wizard® Magnetic 96 DNA Plant System
Cat.#: FF3760 (2 × 96 preps)
FF3761 (4 × 96 preps)
Protocol:
www.promega.com/tbs/tb289/tb289.html
For more information on automated methods visit:
www.promega.com/automethods/
Want to use the Wizard Magnetic 96 DNA Plant
System for high yield or fixed yield gDNA
purification from plant tissue?
Request Application Note #AN105
*Leaf punches 6mm in diameter.
www.promega.com • techserv@promega.com
11
Genomic DNA Purification
Genomic DNA Purification Systems and Accessories
Product
Wizard® Genomic DNA Purification Kit
Size
100 isolations (300µl blood per isolation)
500 isolations (300µl blood per isolation)
100 isolations (10ml blood per isolation)
Cat.#
A1120
A1125
A1620
For Laboratory Use. Cat.# A1120 will give ~40 animal tissue preps, ~80 mouse tail preps, ~80 plant tissue preps, and ~80 cultured cell preps. Please see the Wizard® Genomic DNA Purification Kit
Technical Manual #TM050 for more details and additional supplies necessary for the various preps.
Product
Size
Wizard® SV Genomic DNA Purification System
50 preps (20mg tissue per prep)
250 preps (20mg tissue per prep)
1 each
20 each
Vac-Man® Laboratory Vacuum Manifold, 20-sample capacity
Vacuum Adapters
Cat.#
A2360
A2361
A7231
A1331
The Wizard® SV Genomic DNA Purification System can be used in spin or vacuum format. The Vac-Man® Laboratory Vacuum Manifold can be used to process up to 20 samples at once. The
Vacuum Adapters are required when using the the Vac-Man® Laboratory Vacuum Manifold with the Wizard® SV Genomic DNA Purification System.
Product
Wizard® SV 96 Genomic DNA Purification System
Vac-Man® 96 Vacuum Manifold
Size
1 × 96 preps (20mg tissue per prep)
4 × 96 preps (20mg tissue per prep)
1 each
Cat.#
A2370
A2371
A2291
The Vac-Man® 96 Vacuum Manifold is required for use with the Wizard® SV 96 Genomic DNA Purification System.
Product
MagneSil® ONE, Fixed Yield Blood Genomic System(a)*
MagneSil® Blood Genomic, Max Yield System(a)*
Deep Well MagnaBot® 96 Magnetic Separation Device*
MagnaBot® Spacer, 1/8 inch
Size
1 × 96 preps (50–60µl blood per prep)
1 × 96 preps (200µl blood per prep)
1 each
1 each
Cat.#
MD1370
MD1360
V3031
V8581
* For Laboratory Use. Both MagneSil® Blood Genomic Systems require use of an automated liquid handler such as the Beckman Coulter Biomek® FX. The Deep Well MagnaBot® 96 Magnetic
Separation Device and the MagnaBot® Spacer, 1/8 inch, are also required for use with these systems.
Product
MagneSil® Genomic, Fixed Tissue System
MagneSphere® Technology Magnetic Separation Stands
Size
100 samples
1.5ml (2 position)
1.5ml (12 position)
Cat.#
MD1490
Z5332
Z5342
One time purchase of a magnetic stand required to use product.
Product
Size
MagneSil® KF, Genomic System
Cat.#
200 preps
MD1460
Size
Cat.#
Designed for use with the Thermo Electron KingFisher® mL Instrument.
Product
Wizard® Magnetic 96 DNA Plant System(a)
MagnaBot® 96 Magnetic Separation Device
MagnaBot® Spacer
2 × 96 preps
4 × 96 preps
1 each
1 each
FF3760
FF3761
V8151
V8381
The Wizard® Magnetic DNA Plant System requires use of the MagnaBot® 96 Magnetic Separation Device and the MagnaBot® Spacer for manual or automated DNA purification.
Related Products
Product
Size
Proteinase K
RNase A Solution, 4mg/ml
100mg
1ml
Cat.#
V3021
A7973
For Laboratory Use. Proteinase K is required for tissue preparations. RNase A is certified DNase-free and is used to remove RNA from gDNA preps.
12
Promega DNA Analysis Notebook
Amplifying DNA
Promega offers serveral options for routine, difficult and
high-fidelity PCR.
Taq DNA
Polymerase
Pfu DNA
Polymerase
Tli DNA
Polymerase
PCR Core
Systems
Standard Taq
DNA Polymerase
Routine
Amplification
Everyt
hin
includin g for PCR
g
Availabl dNT Ps.
e with
controls
.
Great
for be
ginners
!
High-fidelity
Amplifications
Difficult
Amplification
GoTaq® DNA
Polymerase
Greater
performan
ce
than stand
ard
Taq Polym
erase
with conv
enience o
f
direct ge
l loading .
www.promega.com • techserv@promega.com
PCR Master
Mix
4375MA11_3A
Higher Performance
Taq DNA Polymerase
r
Greate
ance
perform
ndard
than sta
erase
m
y
l
o
P
q
Ta
venience
n
o
c
s
u
l
p
ter mix.
s
a
m
a
of
be
Can also
t 4°C.
a
d
e
r
o
t
s
13
Amplifying DNA
Overview
Denature… Anneal… Extend… PCR amplification led to a
revolution in molecular biology in the 1980’s. PCR is a
relatively simple technique by which a DNA or cDNA
template is amplified many thousand- or millionfold
quickly and reliably, generating sufficient material for
subsequent analyses.
The PCR process is exquisitely sensitive. While most
biochemical analyses—including nucleic acid detection
with radioisotopes—require the input of significant
amounts of biological material, the PCR process
requires very little starting material. This feature makes
the technique extremely useful, not only in basic
research, but also for applications such as genetic
identity testing, forensic analysis, industrial quality
control and in vitro diagnostics.
The availability of such a powerful tool has led to
significant developments in answering biological
questions. Many adaptations of the original PCR method
have been published, and numerous factors that are
critical for accurate amplification have been identified.
How Much Enzyme is Needed in a Reaction?
Promega recommends using 1.25 units of
thermostable DNA polymerase per 50µl amplification
reaction. For most applications, the enzyme will be in
excess. The inclusion of more enzyme will not
significantly increase yield. Increased amounts of
enzyme and excessively long extension times will
increase the likelihood of artifacts due to the intrinsic
5′→3′ exonuclease activity of Taq DNA Polymerase(e)
and other non-proofreading DNA polymerases.
Artifacts are generally seen as smeared bands in
ethidium bromide-stained agarose gels.
The most frequent cause of excessive enzyme levels
is pipetting error. Accurate dispensing of
submicroliter (<1µl) volumes of enzyme solutions in
50% glycerol is nearly impossible. We strongly
recommend making reaction master mixes sufficient
for the number of reactions being performed. A
master mix increases the volume of pipetted
reagents and reduces pipetting errors.
14
For more
information on
reaction
optimization,
see pp. 16–19.
Typical Reaction with Taq DNA Polymerase.
Nuclease-Free Water
to 50µl final
Reaction Buffer(10X or 5X)
dNTPs
1X
0.2mM each
Taq DNA Polymerase
MgCl2*
1.25u
0.5–4.0mM
Downstream primer
1µM (50pmol)
Upstream primer
1µM (50pmol)
Template
104 copies
*Some reaction buffers contain Mg2+, and additional MgCl2 may not be required.
The optimal Mg2+ concentration depends on the template but is typically in the range
0.5–4mM.
Assemble reactions on ice in the order listed. Be sure to vortex the MgCl2 solution,
primers, dNTPs and Reaction Buffer prior to addition. When using a thermal cycler without
a heated lid, overlay the reaction with 1–2 drops of mineral oil to prevent evaporation.
Setting up reactions with a
proofreading polymerase?
Proofreading enzymes like to digest
free primers due to their 3'→5'
exonuclease activity. Always assemble
the reaction on ice and add the
proofreading polymerase last,
just prior to placing the tubes in a
preheated 94–95°C thermal cycler.
See p. 23 for more information.
ing
-contain
l
C
2
g
M
ll
to use!
r
o
i
Vortex a
r
p
ly
thorough
s
n
o
i
t
u
l
form a
so
n
a
c
s
n
o
soluti
on
MgCl 2
dient up
a
r
g
n
o
i
a
rat
ortex is
concent
v
o
t
e
r
e!
. Failu
R failur
C
thawing
P
f
o
source
common
Promega DNA Analysis Notebook
Amplifying DNA
Example Cycling Conditions for Taq DNA Polymerase.
Step
Initial
Denaturation(a)
Denaturation
Annealing
Extension
Final Extension
Soak
Temperature
Time
(minutes)
95°C
95°C
42–65°C(b)
72°C
72°C
4°C
2
0.5–1
0.5–1
1 min/kb(b,c)
5
indefinite
Want to explore the enzymology of Thermostable
DNA Polymerases more thoroughly?
Cycles
1
}
Go to our online Polymerase Guide at:
www.promega.com/guides/
25–35
1
1
Reactions are placed in a thermal cycler that has been preheated to 95°C. The thermal
cycling protocol has an initial denaturation step where samples are heated at 95°C for
2 minutes to ensure that the target DNA is completely denatured. Initial denaturation of
longer than 2 minutes at 95°C is usually unnecessary and may reduce yield. (Some hot
start polymerases require pre-incubation at 95°C to activate the polymerase prior to the
2-minute denaturation step.)
(b) Annealing temperature should be optimized for each primer set based on the primer
melting temperature (Tm). See section on primer design (p. 16).
(c) The extension time should be at least 1 minute/kilobase of target. Typically, anything
smaller than 1kb uses a 1-minute extension, 2 minutes for >1kb, 3 minutes for >2kb, 4
minutes for >3kb, etc.
(a)
Comparison of Properties for Some Commonly-Used Thermostable DNA Polymerases.
Thermostable DNA Polymerase
Taq/
AmpliTaq Gold®
Vent®
®
®
Characteristic
AmpliTaq
Platinum Taq
Tfl
Tth
(Tli)
>95%
Resulting DNA ends
3′ A
3′ A
3′ A
3′ A
Blunt
5′→3′ exonuclease activity
Yes
Yes
Yes
Yes
No
3′→5′ exonuclease activity
No
No
No
No
Yes
www.promega.com • techserv@promega.com
4431CA
ng
oofreadi
r
P
a
g
n
Usi
se?
Polymera
merases
ly
o
p
g
n
i
d
Proofrea tle slower
lit
work a roofreading
-p
than non s. Be sure to
se
polymera he extension
t
increase least 2
at
time to r kilobase.
pe
minutes
y need
a
m
u
o
y
Also,
s.
re cycle
o
m
3
2
Deep
Vent®
>95%
Blunt
No
Yes
Pfu
Blunt
No
Yes
15
Amplifying DNA
Optimization of PCR
Magnesium Concentration
Magnesium concentration is an important factor to
optimize when performing PCR. The optimal Mg2+
concentration varies depending on the primers,
template, DNA polymerase, dNTP concentration and
other factors. Taq DNA polymerase is the most common
polymerase used for PCR. Taq has an optimal Mg2+
range of 1–4mM MgCl2. Other polymerases may have
different optimal ranges. For example, Tth DNA
polymerase has a narrower optimal range (1.5–2.5mM
MgCl2), Tli DNA Polymerase displays optimal activity at
2–6mM MgCl2, and Pfu DNA Polymerase has an optimal
range of 2–6mM MgSO4. Tfl DNA Polymerase has an
optimal range of 1–4mM Mg2+ but performs better with
MgSO4 than with MgCl2.
When using a pair of PCR primers for the first time, it is
advisable to perform a magnesium titration in 0.5 or
1.0mM increments to determine the optimal Mg2+
concentration. Some primers will amplify equally well at
a number of Mg2+ concentrations, while others may
have very specific Mg2+ concentration requirements.
With too little Mg2+, the polymerase will have poor
activity. With too much Mg2+, nonspecific amplification
can become a problem. Nonspecific PCR products can
appear as a smear on a gel or as distinct bands of
inappropriate size. Too much Mg2+ can also reduce the
fidelity of the DNA polymerase and lead to a higher
error rate.
Taq DNA polymerase is commonly supplied with buffers
containing a fixed concentration of Mg2+ (giving a final
concentration of 1.5mM in the final reaction). Most Taq
DNA polymerase amplifications work well at this Mg2+
concentration, and the reaction can still be optimized by
adding more Mg2+. Pfu DNA polymerase does not have
as great a dependence upon Mg2+ and is most often
supplied with a buffer containing a final concentration of
2mM Mg2+. However, this does not mean that
optimization is unnecessary, and the final concentration
of Mg2+ can be adjusted up to 6mM as needed.
16
Vortex all MgCl2-containing solutions
thoroughly prior to use!
MgCl2 solutions can form a
concentration gradient upon thawing.
Failure to vortex is a common source
of PCR failure!
Primer Design
PCR primers generally range from 15–30 bases long
and are designed to flank the DNA region of interest.
Primers should have 40–60% GC content, and care
should be taken to avoid sequences that might produce
intermolecular or intramolecular secondary structure. To
avoid the production of primer-dimers, the 3′-ends of
the primers should not be complementary. Primerdimers unnecessarily sequester primers away from the
reaction and result in an unwanted polymerase reaction
that competes with the desired PCR product. Avoid
three G or C nucleotides in a row near the 3′-end of the
primer, as this may result in nonspecific primer
annealing, increasing the synthesis of undesirable
products. Ideally, both primers should have nearly
identical melting temperatures (Tm), allowing both
primers to anneal roughly at the same temperature. The
annealing temperature of the reaction is dependent upon
the primer with the lowest melting temperature. For
assistance with calculating the Tm of any primer, a Tm
calculator is provided on the Promega web site at:
www.promega.com/biomath
ur Tm?
o
y
e
t
a
l
calcu
page at:
h
t
Need to
a
M
o
i
's B
romega
math
Go to P romega.com/bio
ul t s
www.p
r n s re s
am retu f e re n t
r
g
o
r
p
The
re e dif fo r Tm
h
t
m
o
r
f
et h o d s o s e l e ct
m
d
e
h
s
n al s
publi
a
c
u
o
Y
io n .
ex am i n e
cal cula t a p rim e r s to n e t h e
Pro m eg a n d d ete r m i r s i n
e
t h e ir Tmy o u r ow n p rim f e r s .
T m o f t re a ctio n buf
n
diff e re
Promega DNA Analysis Notebook
Amplifying DNA
Optimization of PCR
ing
n anneal be
o
i
t
c
a
e
R
ld
ure shou of
t
a
r
e
p
m
te
f the T m
±5°C o primer that
R
the PC lowest T m.
has the
(continued)
Annealing Temperature
Annealing temperature is another factor that may need to
be optimized in PCR. The melting temperature (Tm) of
the PCR primers should be in the range 42–65°C, unless
the primers fall into a special class, such as degenerate
primers, which have lower Tm. Typically the optimal
annealing temperature is ±5°C of the primer with the
lowest Tm. Ideally the Tm of both primers will be similar
so that the optimal annealing temperatures are close. If
the melting temperatures are more than a few degrees
apart, one primer may need to be redesigned so that the
Tm is closer to that of the other primer. A good starting
point is to set the annealing temperature equal to the Tm
of the primers. If nonspecific amplification occurs, this is
a good indication that the annealing temperature needs
to be raised a few degrees. If the PCR reaction yields no
product, this may indicate that the annealing temperature
is too high and should be reduced by several degrees.
Template Quantity
The amount of template required for successful
amplification is dependent upon the complexity of the
DNA sample. For example, in a 4kb plasmid containing
a 1kb insert, 25% of the input DNA is the target of
interest. Conversely, a 1kb gene in the human genome
(3.3 × 109bp) represents approximately 0.00003% of
the input DNA. Approximately 1,000,000-fold more
human genomic DNA is required to maintain the same
number of target copies per reaction. Two common
mistakes are the use of too much plasmid DNA or too
little genomic DNA. If possible, start with up to
104 copies of the target sequence to obtain a signal
in 25–30 cycles, but do not exceed 10ng/µl
(i.e., 500ng/50µl reaction).
Template Quality
The purity and integrity of the DNA template can also be
critical. Obviously there are numerous inhibitors that can
interfere with amplification. These may be copurified
from the original source of the nucleic acid (e.g., the
tissue from which the DNA was isolated). Contaminants
can also be introduced during the purification process.
Examples of common contaminants that can inhibit PCR
are phenol, ethanol, as little as 0.01% SDS or other
detergents, heparin and salts. These contaminants can
usually be removed by a simple phenol:chloroform
extraction followed by ethanol precipitation, or by use of
a PCR clean-up system (see Chapter 3). Some sample
types, such as blood, soil, fungus, plants with high
phenolic content, and fecal samples, are problematic
because they contain strong PCR inhibitors that can be
copurified with the DNA. An easy way to identify
inhibitors in your template nucleic acid is to add an
aliquot of template to the positive control reaction. If
this “spiked” control reaction fails, the template needs to
be further purified before amplification.
Test template quality by adding a control template that
you know amplifies easily and reliably. Combine your
problematic template with 100–1,000 copies of the
control template, and amplify the control template.
Perform the same amplification with the control
template alone. If amplification of the control template
fails only when the problematic template is present,
inhibitors in the problematic template may be to blame.
How M
any
DNA Te Molecules in Yo
ur
mplate?
1µg of
1kb dsD
NA =
9.12 x 1 11
1µg of
0 mole
pGEM ®
cules
Vector
D
2.85 x 1 NA =
10 1 mo
1µg of
lecules
lambda D
NA =
1.9 x 10 10
1µg of
molecule
E. coli
s
genomic
DNA =
2 x 10 8
1µg of
molecule
human g
s
enomic
DNA =
3.04 x 5
10 mole
www.promega.com • techserv@promega.com
cules
17
Amplifying DNA
Betaine, DMSO and formamide can be helpful when
amplifying GC-rich templates. Betaine reduces the
amount of energy required to separate the strands of a
GC-rich DNA template (1). Dimethylsulfoxide (DMSO)
and formamide are thought to aid in the amplification of
GC-rich templates in a similar manner by interfering
with the formation of hydrogen bonds between the two
strands of DNA (2). Some reactions that amplify poorly
in the absence of enhancers will give a strong PCR
product when betaine (1M), DMSO (1–10%), or
formamide (1–5%) are added to the reaction. DMSO
concentrations greater than 10%, and formamide
concentrations greater than 5% will cause inhibition of
Taq DNA polymerase and, presumably, other DNA
polymerases as well (3).
In some cases, general stabilizing agents such as BSA
(0.1mg/ml), gelatin (0.1–1.0%), and nonionic detergents
(0–0.5%) can overcome failure to amplify a region of
DNA. These additives can increase the stability of the
DNA polymerase and may also coat the sides of the PCR
tubes so that reagents are not lost through adsorption
to the tube walls. BSA has also been shown to
overcome the inhibitory effects of melanin on RT-PCR
(4). Nonionic detergents, such as Tween®-20, NP-40,
and Triton® X-100, have the additional benefit of being
able to overcome the inhibitory effects of trace amounts
of strong ionic detergents, such as 0.01% SDS (5).
18
No template
DMSO +
betaine
betaine
DMSO
None
No template
betaine
DMSO
None
PCR Enhancers
bp
1,198 –
350 –
222 –
3825TA08_2A
In some cases it may be helpful to add certain enhancing
agents to a PCR, despite all other attempts to optimize
conditions. Two good examples are the amplification of
GC-rich templates and amplification of templates that
form strong secondary structures, which can cause DNA
polymerases to stall. GC-rich templates can be
problematic due to inefficient separation of the two DNA
strands or because of the tendency of GC-rich primers to
form intermolecular and intramolecular secondary
structures that compete with template annealing. There
are many PCR-enhancing agents that act through a
number of different mechanisms. PCR-enhancing
reagents will not work with all reactions; the beneficial
effects are often template- and primer-specific.
Green
GoTaq® Buffer
Colorless
GoTaq® Buffer
(continued)
DMSO +
betaine
Optimization of PCR
Amplification of a fragment of the human retinoblastoma gene using GoTaq
DNA Polymerase with Colorless GoTaq Reaction Buffer or Green GoTaq
Reaction Buffer with and without the addition of enhancing agents DMSO and
betaine. Amplification reactions contained 500ng human genomic DNA, 0.8µM of each
primer and 1.25u GoTaq DNA Polymerase in a final volume of 50µl. Reactions contained
no additives, 5% DMSO, 1M betaine or 5% DMSO + 1M betaine as indicated. No-template
control reactions were included. Amplification primers and cycling conditions are as
published in Frackman, S. et al. (1998) Betaine and DMSO: Enhancing agents for PCR.
Promega Notes 65, 27–29.
Ammonium ions can make a PCR reaction more tolerant
of nonoptimal conditions. For this reason, some PCR
reagents include 10–20mM (NH4)2SO4. Other PCR
enhancers include glycerol (5–20%), polyethylene glycol
(5–15%), and tetramethyl ammonium chloride (TMAC;
60mM). The effects of these enhancers are very
template- and primer-specific. It may be easier to design
new primers and determine the optimal conditions for
the new primer pair than to do multiple experiments
with some of these less useful enhancers.
References
1. Rees, W., Yager, T.D., Korte, J. and von Hippel, P.H. (1993) Betaine can
eliminate the base pair composition dependence of DNA melting.
Biochemistry 32,137–44.
2. Geiduschek, E.P. and Herskovitz, T.T. (1961) Nonaqueous solutions of DNA.
Reversible and irreversible denaturation in methanol. Arch. Biochem.
Biophys. 95, 114–29.
3. Varadaraj, K. and Skinner, D. (1994) Denaturants or cosolvents improve the
specificity of PCR amplification of a GC-rich DNA using genetically
engineered DNA polymerases. Gene 140, 1–5.
4. Giambernardi, T.A., Rodeck, U. and Klebe, R.J. (1998) Bovine serum
albumin reverses inhibition of RT-PCR by melanin. BioTechniques 25,
564–66.
5. Gelfand, D.H. and White, T.J. (1990) Thermostable DNA polymerase. In:
PCR Protocols: A Guide to Methods and Applications. Innis, M.A., Gelfang,
D.H., Sninsky, J.J., and T.J. White (eds.) Academic Press, San Diego, CA.
pp. 129–41.
Promega DNA Analysis Notebook
Amplifying DNA
Optimization of PCR
(continued)
Troubleshooting
Most troubleshooting of PCR involves evaluating the
possible areas of optimization. Promega has an
extensive PCR troubleshooting guide included in the
PCR Core Systems Technical Bulletin #TB254.
Need a guide to general PCR
optimization and troubleshooting?
Get PCR Core Systems Technical
Bulletin #TB254 online at:
www.promega.com/tbs/tb254/tb254.html
or request a printed copy from your
local Promega representative.
RT-PCR
Looking for information, tips and techniques for
RT-PCR? Request the free RNA Analysis Notebook.
Ask for literature #BR120 from your local Promega
distributor or Promega Representative. Also available
online at: www.promega.com/guides/
www.promega.com • techserv@promega.com
ons?
e questi
r
o
m
e
v
Ha
Promega
Contact l Services:
a
m
Technic
mega.co
o
r
p
@
v
r
techse
serving
s
t
is
t
n
ie
Sc
s.
scientist
19
Amplifying DNA
Routine PCR
PCR Master Mix
PCR Master Mix: Robust, Convenient Amplification
Reaction
Buffer
Taq DNA
Polymerase
dNTPs
Mg2+
Promega’s PCR Master Mix(f,g) is designed for the rapid
and convenient amplification of many common genomic
and cDNA templates (1). PCR Master Mix, formulated as
a 2X solution, offers a single-tube format for PCR setup,
reducing pipetting times, steps and errors as well as
greatly reducing reagent waste. All necessary PCR
components, except for primers and template DNA, are
contained in the Master Mix. Stability is also a key
feature of PCR Master Mix, which can be stored for up to
24 months at 4°C or be put through as many as 20
freeze-thaw cycles without loss of performance. PCR
Master Mix provides 1.25u of Taq DNA Polymerase,
Reaction Buffer, 200µM of each dNTP, and 1.5mM Mg2+
in the final reaction.
3986MA02_3A
Primer
Template
Template Copies per Reaction
1. Denhart, M. and Doraiswamy, V. (2001) Performance advantages designed
into Promega’s PCR Master Mix. Promega Notes 78, 9–12.
Typical Reaction Set-Up with PCR Master Mix.
Template (up to 104 copies of target)
Primers (50pmol each or 1µM final conc.)
Nuclease-Free Water (provided)
PCR Master Mix*
Total Volume
Xµl
Yµl
Zµl
25µl
50µl
10
100
10
100 1,000
M
Detection of low copy number templates using PCR Master MIx. A 360bp
portion of the single-copy α1-antitrypsin gene was amplified from the indicated amounts of
Human Genomic DNA (Cat.# G3041). Lane M, 100bp DNA Ladder (Cat.# G2101).
100
– 360bp
Scalability of PCR Master Mix. The 360bp α1-antitrypsin message was amplified from
10 or 100 copies of Human Genomic DNA (Cat.# G3041) in 10, 25 and 50µl reaction
volumes as indicated.
PCR Master Mix
Cat.#: M7501 (10 reactions)
M7502 (100 reactions)
M7505 (1,000 reactions)
Protocol:
www.promega.com/tbs/9pim750/9pim750.html
Citations detailing use of PCR Master Mix online at:
www.promega.com/citations/
20
10
3441TA06_1B
100
50µl
3278TA03_1A
M 10
25µl
2
10
Reaction Volume
10µl
0
0n
10 g
ng
1n
g
10
0p
10 g
pg
1p
g
* Provides dNTPs (200µM each), Mg2+(1.5mM), and Taq DNA Polymerase (1.25u) at the
final 1X concentration.
M
3063TA09_0A
Reference
PCR Master Mix in two-step RT-PCR. Amplification of a 533bp portion of the
caspase-3 cDNA from 20µl reverse transcription reaction. Reverse transcription was
performed using the ImProm-II™ Reverse Transcription System (Cat.# A3800) and the
indicated amount of total RNA. The cDNA in the entire 20µl reaction was amplified by
adding 15µl of PCR Master Mix, 2µl of gene-specific primers and 13µl of Nuclease-Free
Water (Cat.# P1195). Further details of the experiment may be found in Miller, K., Moravec,
R. and Riss, T. (2001) An integrated approach to studying apoptosis: From gene expression
to cellular events. Cell Notes 2, 4–6.
Cell Notes and
Promega Notes
are available on
line at:
www.promega.c
om
or upon request.
Promega DNA Analysis Notebook
Amplifying DNA
Routine PCR
(continued)
GoTaq® DNA Polymerase:
Direct-to-Gel Amplification
GoTaq® DNA Polymerase
Cat.#: M3001 (100u; 80 reactions)
M3005 (500u; 400 reactions)
M3008 (2,500u; 2,000 reactions)
Supplied with enzyme (5u/µl), 5X Green GoTaq
Reaction Buffer and 5X Colorless GoTaq Reaction
Buffer. Sufficient to give the indicated number of 50µl
reactions using 1.25u of enzyme per reaction.
Protocol:
www.promega.com/tbs/9pim300/9pim300.html
Separation of the components of the GoTaq Green Reaction Buffer during
electrophoresis. PCR samples amplified using GoTaq DNA Polymerase and GoTaq Green
Reaction Buffer were loaded onto an agarose gel. Samples are shown before (A) and after (B)
electrophoresis. Volumes indicate the amount of amplification reaction loaded on the gel.
360bp
1.1kb
1.8kb
2.4kb
3.1kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19 20 M
bp
4,000 –
3,000 ––––––
2,000 –
1,500 –
1,000 –
500 –
250 –
3824TA08_2A
GoTaq DNA Polymerase(e,g) contains native Taq DNA
Polymerase in a proprietary formulation. The GoTaq
enzyme is supplied with 5X Green and 5X Colorless GoTaq
Reaction Buffers. The Green Reaction Buffer contains a
compound that increases sample density so that samples
sink easily into the wells of an agarose gel. The Green
Reaction Buffer also contains two dyes (a blue dye and a
yellow dye) that separate during electrophoresis and can
be used to monitor migration progress. This allows
reactions to be directly loaded onto agarose gels without
the need for loading dye. The blue dye migrates at the
same rate as 3–5kb DNA fragments in a 1% agarose gel.
The yellow dye migrates at a rate faster than primers
(<50bp) in a 1% agarose gel. The Colorless GoTaq
Reaction Buffer has the same formulation as the Green
Reaction Buffer but does not contain dyes. The Colorless
Reaction Buffer is recommended for any application where
absorbance or fluorescence measurements of the PCR
amplimer are necessary before clean-up. Both 5X buffers
are supplied at pH 8.5 and contain MgCl2 at a
concentration of 7.5mM, giving a final concentration of
1.5mM in the reaction.
Amplification of various templates with GoTaq DNA Polymerase and other
Promega Taq DNA Polymerase formulations. The reactions for each template are
loaded in this order: Taq DNA Polymerase in Storage Buffer B (Cat.# M1661); Taq DNA
Polymerase in Storage Buffer A (Cat.# M1861); GoTaq DNA Polymerase in Colorless
Reaction Buffer; GoTaq DNA Polymerase in Green Reaction Buffer. Reactions without the
Green GoTaq Reaction Buffer require the addition of loading dye prior to electrophoresis.
Compatibility of GoTaq DNA Polymerase with Upstream and Downstream Applications.
Product
Cat.# Green Reaction Buffer Colorless Reaction Buffer
T-Vector Cloning
Yes
Yes
pGEM®-T & pGEM®-T Easy Systems(h,i) A1360, A1380, A3600, A3610
pTARGET™ Mammalian Expression Vector System(i,j)
A1410
Yes
Yes
PCR Clean-Up
A1930
Yes
Yes
Wizard® MagneSil® PCR Clean-Up System(a)
A9340
Yes
Yes
Wizard® SV 96 PCR Clean-Up System
A9281
Yes
Yes
Wizard® SV Gel and PCR Clean-Up System
Wizard® PCR Preps DNA Purification System(k)
A7170
Yes
Yes
Two-Step RT-PCR
A3500
Yes
Yes
Reverse Transcription System(l,m)
ImProm-II™ Reverse Transcription System(l,m)
A3800
Yes
Yes
Transcription/Translation
TNT® T7 Quick for PCR DNA(l,m,n)
L5540
Yes
Yes
www.promega.com • techserv@promega.com
21
Amplifying DNA
(continued)
Taq DNA Polymerase & PCR Core Systems:
Value and Quality
Promega is a premier supplier of native Taq DNA
Polymerase. We offer many options for your needs. You
can assemble your own reagents from separate Taq
DNA Polymerase and dNTPs, or purchase the PCR Core
Systems to get everything together in one package. The
PCR Core Systems(f) are supplied with a Technical
Bulletin that contains thorough coverage of
considerations involved in routine PCR amplification and
extensive troubleshooting information.
Taq DNA Polymerase in Storage Buffer A
Cat.#: M1861 (100u; 80 reactions)
M1865 (500u; 400 reactions)
M1868 (2,500u; 2,000 reactions)
Taq DNA
Polymerase
All components
supplied in
PCR Core Systems
dNTPs
10X Thermophilic
Reaction Buffer
MgCl2
See
Primers
Template
p. 25
for d
NT Ps
.
3987MA02_3A
Routine PCR
Supplied with Taq DNA Polymerase 10X Reaction
Buffer, 25mM MgCl2. One reaction uses 1.25u of
enzyme.
Taq DNA Polymerase in Storage Buffer B
Cat.#: M1661 (100u; 80 reactions)
M1665 (500u; 400 reactions)
M1668 (2,500u; 2,000 reactions)
Supplied with Taq DNA Polymerase 10X Reaction
Buffer, 25mM MgCl2. One reaction uses 1.25u of
enzyme.
Citations for use of Taq DNA Polymerase online at:
www.promega.com/citations/
The
PCR
Syste
Core
ms ar
e gre
at for
resea
rcher
s just
learni
ng PC
R
See p. 14 for
typical reaction
set-up.
Taq DNA Polymerase in Storage Buffer A & B
Promega first offered Taq DNA Polymerase stabilized
with Triton® X-100 (Taq DNA Polymerase in Storage
Buffer A). Later we developed Taq DNA Polymerase
stabilized with Tween®-20 and NP-40 (Taq DNA
Polymerase in Storage Buffer B). In most cases there is
no difference in performance. One key distinction
between the two products is compatibility with other
suppliers reaction buffers. Taq DNA Polymerase in
Storage Buffer A must be used with the supplied
Reaction Buffer, which contains 0.1% Triton® X-100 at
the 1X concentration. Taq DNA Polymerase in Storage
Buffer B does not have this requirement and can be
used either with the supplied Promega Reaction Buffer
or with other Taq DNA polymerase reaction buffers.
PCR Core System I
Cat.#: M7660 (200 × 50µl reactions; 1.25u Taq DNA
Polymerase/reaction)
Comes with 250u Taq DNA Polymerase in Storage
Buffer B, Taq DNA Polymerase 10X Reaction Buffer
without MgCl2, Taq DNA Polymerase 10X Reaction
Buffer with MgCl2 (1.5mM at 1X), 25mM MgCl2, PCR
Nucleotide Mix.
PCR Core System II
Cat.#: M7665 (200 reactions; 1.25u Taq
Polymerase/50µl reaction)
Same components as M7660 plus Positive Control
Plasmid DNA template and Upstream and Downstream
Control Primers.
Protocol:
www.promega.com/tbs/tb254/tb254.html
22
Promega DNA Analysis Notebook
Amplifying DNA
Proofreading Polymerases
8
bp
M
Promega
30
B.
3 0.3 0.03 0.0
M
Supplier A
30
3 0.3 0.03 0.0
1,500 –
1,000 –
– 1.2kb
product
100 –
2355TA08_8A
500 –
Comparison of sources of native Pfu DNA Polymerase. A 1.2kb fragment of human
α1-antitrypsin gene was amplified using Pfu DNA Polymerase from Promega (Panel A) and
from another supplier (Panel B). The target was amplified from decreasing amounts of Human
Genomic DNA (Cat.# G3041) as indicated. Lane M, 100bp DNA Ladder (Cat.# G2101).
Pfu DNA Polymerase
Cat.#: M7741 (100u; 80 reactions)
M7745 (500u; 400 reactions)
Each provided with enzyme (5u/µl), Pfu DNA
Polymerase 10X Reaction Buffer (2mM MgSO4 @ 1X)
sufficient to give the indicated number of 50µl
reactions using 1.25u of enzyme.
Protocol:
www.promega.com/tbs/9pim774/9pim774.html
Citations for use of Pfu DNA Polymerase online at:
www.promega.com/citations/
Not available in North America.
7
Accuracy × 105
6
5
4
3
2
1
0
Pfu
Tli
Taq
DNA Polymerase
3973MA02_3A
Incorporation fidelity can be an important consideration
for cloning projects. Thermostable enzymes with a
3′→5′ exonuclease activity, commonly known as
proofreading activity, offer the highest fidelity in
amplification reactions. Proofreading enzymes like
Pfu and Tli DNA Polymerase(e) offer three- to sixfold
higher fidelity than standard Taq DNA Polymerase. In
general, proofreading enzymes extend primers a little
slower than Taq DNA Polymerase and thus typically
require longer extension times and a few more cycles.
Assume 2 minutes per kilobase of amplimer, and add
2–3 cycles to your reaction when using a proofreading
enzyme. With Pfu DNA Polymerase(e), it is important to
use the Reaction Buffer supplied with the enzyme for
maximum fidelity. Pfu Reaction Buffer is formulated to
give maximum fidelity, not maximum yield. After all, you
use Pfu for fidelity not yield. If you need greater yield,
use more template DNA.
A.
se
: Increa
t
n
i
H
e
Usag
extension
n
io
t
a
c
i
f
ampli
standard
r
e
v
o
X
times 2 olymerase
P
Taq DNA kb)
in/
(e.g ., 2m 3 cycles.
2–
and add
Accuracy of thermostable polymerases. The accuracy of Pfu DNA Polymerase has
been reported by Cline et al. as 7.7 × 105. Using the PCR-based forward mutation assay,
they reported the accuracy of Pfu as approximately two-fold higher than Vent® (Tli DNA
Polymerase) and approximately sixfold higher than Taq DNA Polymerase (1).
Reference
1. Cline, J., Braman, J.C. and Hogrefe, H.H. (1996) PCR fidelity of Pfu DNA
polymerase and other thermostable DNA polymerases. Nucl. Acids Res. 24,
3546–51.
ave a 3′→5′
Proofreaders h
in
that is lacking
ty
vi
ti
ac
e
as
le
exonuc
Taq
g enzymes like
non-proofreadin
ymes
roofreading enz
P
.
se
ra
e
m
ly
Po
DNA
the reaction is
if
rs
e
im
pr
e
d
can degra
too long prior to
r
fo
t
si
to
d
e
allow
mend setting up
om
c
re
e
W
n.
io
amplificat
and adding the
e
ic
on
s
on
ti
reac
ior to
lymerase just pr
proofreading po
eated
tion into a preh
ac
re
e
th
g
in
plac
thermal cycler.
Tli DNA Polymerase
Cat.#: M7101 (50u; 40 reactions)
Supplied with enzyme (5u/µl), Thermophilic DNA
Polymerase 10X Reaction Buffer and 25mM MgCl2.
Sufficient to give the indicated number of 50µl
reactions using 1.25u of enzyme/reaction.
Citations for use of Tli DNA Polymerase online at:
www.promega.com/citations/
www.promega.com • techserv@promega.com
23
Amplifying DNA
Hot start PCR is a commonly used technique to reduce
nonspecific amplification. One cause of nonspecific
amplification is the assembly of PCR reactions at room
temperature or on ice. Under these conditions, the PCR
primers may be able to anneal to various noncomplementary positions on the template. Although
activity of thermostable DNA polymerases at room
temperature or 4°C is usually less than 25%, they can
extend nonspecifically annealed primers at these
temperatures. Any newly synthesized product is
completely complementary to the PCR primer, allowing
the primer to anneal specifically to this region during
PCR, resulting in an undesired amplification product.
Hot start PCR can also reduce the amount of primerdimer formed. Primer-dimers result from
complementarity between the 3′ ends of the PCR primers.
At room temperature or on ice, these complementary
regions anneal and the polymerase extends the ends to
produce a primer-dimer. Primer-dimers often appear as a
diffuse band at ~50–100bp on ethidium bromide-stained
gels. Both nonspecific products and primer-dimers can
compete with the desired amplification reaction for
reagents. By avoiding the conditions that lead to
nonspecific amplification, hot start PCR can improve the
yield of the desired PCR product.
There are several ways to perform hot start PCR. The
reaction can be assembled on ice or at room
temperature, omitting the DNA polymerase until the
reaction has been placed in the thermal cycler and
heated to 60–65°C. Once the reaction has reached
60–65°C the desired amount of polymerase can be
added. This prevents the polymerase from extending
primers until the higher temperature is reached and
primer annealing is more specific. The method is quite
effective but can be labor-intensive, particularly if
dozens of amplification reactions are involved.
Another approach to hot start PCR involves the use of
wax to physically sequester one or more critical reaction
TaqBead™ Hot Start Polymerase
Cat.#: M5661 (100 reactions)
Supplied with 100 beads (1.25u Taq DNA Polymerase
in Storage Buffer B/bead), Thermophilic DNA
Polymerase 10X Reaction Buffer, and 25mM MgCl2.
Protocol:
www.promega.com/tbs/tb247/tb247.html
Citations for use of TaqBead Polymerase online at:
www.promega.com/citations/
24
components until the appropriate temperature is
reached. Wax beads can be added to a PCR before the
addition of the DNA polymerase. Heating the PCR to
60°C melts the wax, which forms a liquid layer over the
surface of the reaction, eliminating the need for mineral
oil. Upon cooling to 4°C, the wax solidifies. The DNA
polymerase is added onto this wax layer and, as the
PCR is heated during the first denaturation step, the wax
melts and the polymerase can access the other PCR
reagents. This method is labor intensive—requiring an
additional heating and cooling step to prepare the wax
layer. Also, opening the PCR tube to add the polymerase
increases the risk of contamination. Additionally, the
solid wax layer that forms upon cooling to 4°C will clog
pipet tips when attempting to break through the wax to
pipet the PCR. Thus, it is often necessary to use one
pipet tip to puncture the wax layer and a second pipet
tip to remove the PCR products.
Taq Bead Hot Start Polymerase
Enter Promega’s Taq Bead Hot Start Polymerase(f). By
impregnating a wax bead with Taq DNA Polymerase, the
additional heating and cooling steps to form the wax
layer are eliminated. A single bead is added to each 50µl
reaction, and as the reaction is heated, the wax melts
and releases the polymerase. The molten wax rises to
the surface of the PCR where it forms an incomplete
barrier. In thermal cyclers with heated lids, a hole
remains above the reaction to ease pipetting. To prevent
evaporation in thermal cyclers without heated lids, we
recommend adding mineral oil to each PCR. The molten
wax and mineral oil will mix during the thermal cycling
to form a single layer, which solidifies when the reaction
is cooled to 4°C and can clog pipet tips.
Taq
Cold Start
M
10
1
0.1
TaqBead™
Hot Start
10
1
0.1
pg of
target
bp
2,645 –
1,605 –
1,198 –
– 1.8kb
676 –
517 –
396 –
1586TA09_6A
Hot Start Methodology
– primerdimer
Hot start amplification reduces the yield of nonspecific amplification products.
Aliquots of 10, 1 or 0.1pg pGEM®-luc Vector(h,o) (Cat.# E1541) were diluted in 30ng of
Human Genomic DNA (Cat.# G3041). A 1.8kb luciferase gene product was amplified by
PCR using Taq DNA Polymerase (Storage Buffer B) or TaqBead Hot Start Polymerase in
Promega Reaction Buffer supplemented with 2mM MgCl2. Details are provided in Miller, K.,
Smith, R. and Storts, D. (1996) Improved PCR amplification using TaqBead Hot Start
Polymerase. Promega Notes 60, 2–6.
Promega DNA Analysis Notebook
Amplifying DNA
dNTPs
1
2
3
4
5
6
7
Promega is a premier supplier of high-quality dNTPs(f)
in bulk form or premixed in the PCR Nucleotide Mix(f).
Promega’s dNTPs are >99% triphosphate with verified
concentrations. All dNTPs, whether bulk or premixed,
are DNase- and RNase-free, and are functionally tested
in amplification reactions. The PCR Nucleotide Mix is
also functionally tested in RT-PCR.
3528TA08_1A
Set of dATP, dCTP, dGTP and dTTP
Cat.#: U1330 (10µmol each; 1,000 reactions)
U1420 (25µmol each; 2,500 reactions)
U1240 (40µmol each; 4,000 reactions)
U1410 (200µmol each; 20,000 reactions)
RT-PCR functional assay using PCR Nucleotide Mix. The dNTPs were used following
50 freeze-thaw cycles. Amounts of template RNA: lane 1, 25fmol; lane 2, 2.5fmol; lane 3,
250amol; lane 4, 25amol; lane 5, 2.5amol; lane 6, 250zmol; lane 7, no template control.
Set of dUTP, dATP, dCTP and dGTP
Cat.#: U1335 (10µmol each; 1,000 reactions)
U1245 (40µmol each; 4,000 reactions)
PCR Nucleotide Mix
Each dNTP is supplied at 100mM. Reaction size is
considered to be 200µM each dNTP in a 50µl PCR
reaction.
Custom and bulk dNTP sizes are available.
bp
1,500–
1
2
3
4
5
6
Cat.#: C1141 (200µl; 200 reactions)
C1145 (1,000µl; 1,000 reactions)
The PCR Nucleotide Mix supplies a single solution
containing each dNTP (dATP, dTTP, dGTP, dCTP) at
10mM. Reaction size is considered to be 200µM of
each dNTP in a 50µl reaction. Each reaction uses 1µl
of PCR Nucleotide Mix.
Custom and bulk PCR Nucleotide Mix sizes are
available.
Protocol:
www.promega.com/tbs/9pic114/9pic114.html
1,000–
3527TA08_1A
500–
Stability of dNTPs. The integrity of the RT-PCR products on the gel demonstrates the
stability of the dNTPs after storage under the conditions listed. The dNTPs used in each
RT-PCR were stored as follows prior to use: Lane 2, 1 freeze-thaw cycle; lane 3, 50 freezethaw cycles; lane 4, 1 year at –20°C; lane 5, negative PCR control; lanes 1 and 6, 100bp
DNA Ladder (Cat.# G2101).
www.promega.com • techserv@promega.com
Ps and
T
N
d
’s
a
ix
Promeg
otide M
le
c
u
N
PCR
at least
h
g
u
o
r
h
t
can go
.
e/thaws
z
e
e
r
f
50
25
Amplifying DNA
Routine PCR
Product
PCR Master Mix(f,g)
Size
Cat.#
Size
Cat.#
100 reactions
1,000 reactions
M7502
M7505
For Laboratory Use. A reaction consists of 25µl of the 2X PCR Master Mix in a 50µl total volume. Supplied with Nuclease-Free Water.
Product
GoTaq® DNA Polymerase(e,g)
100u
500u
2,500u
M3001
M3005
M3008
For Laboratory Use. Supplied with 5X Green GoTaq® Reaction Buffer and 5X Colorless GoTaq® Reaction Buffer. Both buffers contain 1.5mM Mg2+ at the 1X concentration.
Product
PCR Core System I(f)
PCR Core System II(f)
Size
200 reactions
200 reactions
Cat.#
M7660
M7665
For Laboratory Use. PCR Core Systems provide Taq DNA Polymerase in Storage Buffer B, PCR Nucleotide Mix, Taq DNA Polymerase 10X Reaction Buffers with and without MgCl2, and 25mM
MgCl2 sufficient for 200 × 50µl reactions containing 1.25u of Taq DNA Polymerase. PCR Core System II also contains Positive Control Plasmid DNA template, and Upstream and Downstream
Control Primers.
Product
Taq DNA Polymerase in Storage Buffer A(e)
(Supplied with Taq DNA Polymerase 10X Reaction Buffer without MgCl2, and 25mM MgCl2.)
Taq DNA Polymerase in Storage Buffer A(e)
(Supplied with Taq DNA Polymerase 10X Reaction Buffer with MgCl2, giving 1.5mM Mg2+ at the 1X concentration.)
Taq DNA Polymerase in Storage Buffer B(e)
(Supplied with Taq DNA Polymerase 10X Reaction Buffer without MgCl2, and 25mM MgCl2 Solution.)
Taq DNA Polymerase in Storage Buffer B(e)
(Supplied with Taq DNA Polymerase 10X Reaction Buffer with MgCl2, giving 1.5mM Mg2+ at the 1X concentration.)
Size
Cat.#
Size
Cat.#
50u
M7101
100u
500u
2,500u
100u
500u
2,500u
100u
500u
2,500u
100u
500u
2,500u
M1861
M1865
M1868
M2861
M2865
M2868
M1661
M1665
M1668
M2661
M2665
M2668
For Laboratory Use.
Proofreading Polymerases
Product
Pfu DNA Polymerase(e)*
(Supplied with Pfu DNA Polymerase 10X Reaction Buffer with MgSO4, giving 2mM Mg2+ at the 1X concentration.)
100u
500u
M7741
M7745
Tli DNA Polymerase(e)**
(Supplied with Thermophilic DNA Polymerase 10X Reaction Buffer and 25mM MgCl2.)
*Not Available in North America. **For Laboratory Use.
26
Promega DNA Analysis Notebook
Amplifying DNA
Hot Start Polymerase
Product
TaqBead™ Hot Start Polymerase(f)
(Supplied with Thermophilic DNA Polymerase 10X Reaction Buffer and 25mM MgCl2.)
For Laboratory Use. One bead per reaction; 1.25u Taq DNA Polymerase per bead.
Size
Cat.#
100 reactions
M5661
Size
Cat.#
Size
Cat.#
dNTPs
Product
PCR Nucleotide Mix(f)
200µl
1,000µl
10µmol
25µmol
40µmol
200µmol
10µmol
40µmol
(Contains 10mM each dNTP; use 1µl per 50µl reaction.)
Set of dATP, dCTP, dGTP, and dTTP(f)
(100mM each dNTP. Individual tubes available.)
Set of dUTP, dCTP, dGTP, and dATP(f,p)
(100mM each dNTP.)
For Laboratory Use.
C1141
C1145
U1330
U1420
U1240
U1410
U1335
U1245
Accessories
Product
Promega 10 Barrier Tips, 960/pk
Promega 10E Barrier Tips, 960/pk
Promega 10F Barrier Tips, 960/pk
Promega 20 Barrier Tips, 960/pk
Promega 100 Barrier Tips, 960/pk
Promega 200 Barrier Tips, 960/pk
Promega 1000 Barrier Tips, 480/pk
Mineral Oil*
Nuclease-Free Water*
0.5–10µl
0.5–10µl
0.5–10µl
2–20µl
10–100µl
50–200µl
100–1,000µl
12ml
150ml
A1491
A1501
A1511
A1521
A1541
A1551
A1561
DY1151
P1195
*For Laboratory Use.
Tip and Pipette Compatibility Guide
Promega 10
Promega 10E
Promega 10F
Promega 20
Promega 100
Promega 200
Promega 1000
Size
0.5–10µl
0.5–10µl
0.5–10µl
2–20µl
10–100µl
50–200µl
100–1,000µl
Pipetman®
P-2; P-10
P-2; P-10
Eppendorf®
0.5–10µl
Oxford
Benchmate®
0.5–10µl
0.5–10µl
Finnpipette®
0.5–10µl Digital
0.5–10µl
P-20
P-100
P-200
P-1000
www.promega.com • techserv@promega.com
2–20µl
10–100µl
EDP–250µl
10–50µl
40–200µl
200–1,000µl
5–40µl
40–200µl
200–1,000µl
27
PCR Clean-Up
Overview
Downstream applications such as T-Vector cloning,
restriction digestion and direct sequencing benefit from
clean-up of PCR amplimers. T-vector cloning has a
tremendous dependence upon PCR product purity.
Although unpurified amplification reactions can be used
for T-vector cloning, more screening of the resulting
colonies is generally necessary to find the specific clone
of interest. This is because unpurified PCR amplification
reactions can contain primer-dimers and nonspecific
amplimers in much higher molar quantities than the PCR
product of interest. These nonspecific products compete
for ligation with the amplimer of interest. Typically, an
experiment with unpurified products will produce a large
number of colonies, many of which contain small,
nonspecific PCR products as inserts. Thus, the efficiency
of the cloning experiment is reduced. In one case the
percentage of colonies containing the correct insert was
67% using unpurified PCR products. In contrast, when
purified PCR products were used >90% of colonies
contained the correct insert (1).
Gel Isolation
Gel isolation is the most effective way to isolate the PCR
product you need for your downstream applications.
Agarose gel electophoresis allows you to separate the
desired amplimer from any nonspecific bands, primers
and primer-dimers. You visualize your gel quickly on a
UV lightbox, using 10mg wavelength UV light, cut out
the band of interest and then purify the product. Gel
isolation is typically used if the downstream application
is cloning and additional bands, representing
nonspecific amplimers or primer-dimer, are present on
the gel. The agarose is melted and combined with a
chaotrope like guanidine, and the DNA is then bound to
silica. Agarose and guanidine are quickly and efficiently
removed by an alcohol wash, and the purified DNA is
eluted in water.
Direct Isolation
Many downstream applications such as DNA sequencing
and single nucleotide polymorphism (SNP) analysis
require that salts, dNTPs, proteins and primers be
removed from the amplification product as they can
interfere with further enzymatic reactions. Many
researchers use a simple ethanol precipitation prior to
sequencing. This removes most dNTPs and salts but
leaves behind protein and may also leave primers.
Phenol:chloroform extraction can remove protein
contaminants but recovery rates can be low, and the use
of organics is undesirable. Use of silica technology
simplifies the whole process. The amplification reaction
products are bound to silica in the presence of a
chaotrope like guanidine. Salts, dNTPs and primers pass
by the silica. Primer-dimers and nonspecific amplimers
smaller than 70bp are not bound efficiently. A simple
alcohol wash removes the guanidine and protein while
material >70bp is retained. The purified DNA is eluted in
water or another low-ionic strength solution. The
success of the downstream application is dependent
upon the specificity of the amplification reaction, as any
nonspecific amplimers will be copurified with the
specific product of interest.
Reference
1. Buros, M. and Betz, N. (2002) Removal of ethidium bromide and calf
intestinal alkaline phosphatase using the Wizard® SV Gel and PCR
Clean-Up System. eNotes:
www.promega.com/enotes/applications/ap0045_tabs.htm
PCR Reaction
Gel Purification
Direct Purification
Wizard® SV
Gel & PCR
Clean-Up System
Wizard® PCR
Preps DNA
Purification
System
Wizard®
MagneSil® PCR
Clean-Up System
28
4020MA03_3A
Wizard® SV 96
PCR Clean-Up
System
Promega DNA Analysis Notebook
PCR Clean-Up
100bp
U
Wizard® SV Gel and PCR Clean-Up System
Cat.#:
A9281 (50 preps)
A9282 (250 preps)
Protocol:
www.promega.com/tbs/tb308/tb308.html
P
P
U
P
1,000bp
P
P
P
500bp
P
U
P
100bp
U
P
500bp
U
P
1,000bp
U
P
3,199bp
U
P
P
U
P
P
P
Gel analysis of PCR products before and after direct purification using the
Wizard SV Gel and PCR Clean-Up System. DNA fragments of the sizes indicated
were analyzed before (U) and after (P) direct purification from amplification reactions.
100
90
80
70
60
50
40
30
20
10
0
10
15
25
50
75
3789TA07_2A
1,000bp –
P: Purified
Gel analysis of PCR products before and after gel extraction using the Wizard
SV Gel and PCR Clean-Up System. Recovery of various sizes of unpurified (U) and
purified (P) PCR products. Purified products were extracted from a 1% agarose gel run with
TAE buffer. Lane M, 1kb DNA Ladder (Cat.# G5711).
ig
as b
A
N
ar D
e
n be
n
i
a
L
c
0kb
up to
as 1
h
t
i
ed w very.
i
f
i
r
pu
reco
%
5
9
www.promega.com • techserv@promega.com
100
Elution volume versus recovery for a 700bp PCR product purified directly
from an amplification reaction using the Wizard SV Gel and PCR Clean-Up
System. One hundred percent represents recovery with 50µl elution volume. Adapted
from Table 4 in Betz, N. and Strader, T. (2002) Clean up with Wizard SV for Gel and PCR.
Promega Notes 82, 2–5.
3,000bp –
U: Unpurified
P
1,500bp
Elution Volume (µl)
M
P
3790TB08_2A
U
Percent Recovery
The Wizard SV Gel and PCR Clean-Up System is
designed to extract and purify DNA fragments directly
from PCR reactions or from agarose gels. Fragments of
100bp–10kb can be recovered from standard or lowmelt agarose gels in either Tris acetate (TAE) or Tris
borate (TBE) buffer. Up to 95% recovery is achieved,
depending upon the DNA fragment size. This
membrane-based system, which can bind up to 40µg of
DNA, allows recovery of isolated DNA fragments or PCR
products in as little as 15 minutes, depending on the
number of samples processed and the protocol used.
Samples can be eluted in as little as 15µl of NucleaseFree Water. The purified DNA can be used for automated
fluorescent sequencing, cloning, labeling, restriction
enzyme digestion or in vitro transcription/translation
without further manipulation.
P
200bp
3972MA02_3A
Wizard® SV Gel and PCR
Clean-Up System
Effect of Various PCR Additives on Recovery of a 1,000bp
PCR Product Using the Wizard SV Gel and PCR Clean-Up
System Direct Purification Method.
Percent Recovery
PCR Additive
Relative to “No Additive”
No Additive
100%
1M Betaine
94%
1M Q-Solution
97%
0.1% Triton® X-100
92%
0.1% Tween® 20
87%
0.1% NP-40
82%
5% Glycerol
87%
5% Formamide
90%
5% DMSO
87%
0.5M Tetramethylene Sulfoxide
94%
0.4M Sulfolane
94%
0.4M 2-Pyrolidene
95%
1mM Tartrazine
100%
1% Ficoll®-400
100%
29
PCR Clean-Up
If it
work
s in
it’ll
SV,
work
in S
V9
6.
Wizard® SV 96
PCR Clean-Up System
Recommended
automated system
for reactions prepared
in the presence of
PCR enhancers like
betaine, DMSO, etc.
Just like the SV
system, SV 96 works
with a wide variety
of PCR additives.
Manu
al or
a
96-w utomated
ell P
purific CR
ation.
1M Betaine
Standard PCR
3445CA06_1A
The Wizard SV 96 PCR Clean-Up System provides a
fast, simple technique for the efficient isolation of
purified DNA fragments generated by PCR amplification.
Walkaway automation is easily achieved on any 96-well
liquid handling workstation equipped with a gripper and
vacuum apparatus. Double-stranded DNA fragments can
be purified from 96 samples in less than 20 minutes.
Purification is achieved without phenol:choroform
extraction or ethanol precipitation. Optimized methods
are available for the Beckman Coulter Biomek® 2000 and
FX instruments.
Wizard® SV 96 PCR Clean-Up System
Cat.#: A9340 (1 × 96 preps)
A9341 (4 × 96 preps)
A9342 (8 × 96 preps)
Protocol:
www.promega.com/tbs/tb311/tb311.html
For information on automated methods, visit:
www.promega.com/automethods/
Want to do 96-well agarose gel isolations?
3796TB09_2A
Request Application Note #AN096.
Microarray of purified PCR products. Representative microarray blocks of PCR
product purified using the Wizard SV 96 PCR Clean-Up System and hybridized to
complementary Cy®3-labeled cDNA. PCR DNA was isolated from a standard amplification
reaction or from a reaction containing 1M betaine. No effect of betaine is observed.
100bp
200bp
300bp
500bp
P
P
P
P
U
U
U
U
1,000bp
P
U
3801TA08_2A
M
Agarose gel analysis of PCR fragments purified on the Beckman Coulter
Biomek® 2000 liquid handler. PCR fragments of 100, 200, 300, 500 and 1,000bp were
purified using the Wizard SV 96 PCR Clean-Up System on the Beckman Coulter Biomek®
2000 robotic workstation. Both purified (P) and unpurified (U) fragments were separated on
an ethidium bromide-stained 2% agarose gel. Percent recovery ranged from 71 ± 3% for
the 100bp fragment to 100 ± 1% for the 1,000bp fragment. Lane M, 100bp DNA Ladder
(Cat.# G2101).
30
Promega DNA Analysis Notebook
PCR Clean-Up
Wizard® MagneSil®
PCR Clean-Up System
Wizard® MagneSil® PCR Clean-Up System
The MagneSil PCR Clean-Up procedure is fast and
reliable. PCR products bind to MagneSil particles in the
presence of guanidine hydrochloride and remain tightly
bound during washing. Purified DNA is eluted in water
and may be used for automated fluorescent sequencing
and microarray spotting. The MagneSil PCR Clean-Up
System is ideally suited for reactions prepared using
PCR Master Mix and GoTaq® DNA Polymerase. It also
works well with reactions prepared using AmpliTaq® and
AmpliTaq Gold® DNA Polymerase.
For information on automated methods visit:
www.promega.com/automethods/
100
80
60
PCR Master Mix
40
AmpliTaq® DNA
Polymerase
20
0
0
200
400
600
800 1,000
PCR Product Size (bp)
3289MA03_1A
PCR clean-up is performed using Promega’s unique
MagneSil Paramagnetic Particles(a) and the MagnaBot® 96
Magnetic Separation Device (Cat.# V8151) fitted with a
3/16" MagnaBot Spacer (Cat.# V8381). The MagnaBot 96
Magnetic Separation Device is designed to work with
most robotic platforms. A MagnaBot 384 Magnetic
Separation Device is also available (Cat.# V8241).
Protocols:
Automated 96-well plate protocol
www.promega.com/tbs/tb290/tb290.html
Automated 384-well plate protocol
www.promega.com/tbs/ep009/ep009.html
% Recovery
The Wizard MagneSil PCR Clean-Up System(a) removes
impurities from PCR reactions, giving high quality and
yield of double-stranded amplicons. The system
removes excess nucleotides, primers and small
amplification products such as primer-dimers from PCR
reactions. The system is designed for automation on
laboratory robotic systems for unattended 96- to
384-well purification.
Cat.#: A1930 (4 × 96 preps)
A1931 (8 × 96 preps)
A1935 (100 × 96 preps)
Percent recovery of PCR products purified using the Wizard MagneSil PCR
Clean-Up System. PCR amplimers were purified from standard amplification reactions
performed using either PCR Master Mix or AmpliTaq® DNA Polymerase.
Cy®3
Cy®5
3578TA11_1A
E. coli Control Array
Array images after hybridization with Cy3 and Cy5 fluorescent control
E. coli targets. E. coli genomic DNA was amplified using 96 unique primer pairs. The
Wizard MagneSil PCR Clean-Up System-purified PCR products were printed onto a
poly-L-lysine-coated slide and hybridized to Cy3- or Cy5-labeled E. coli cDNA. For further
details, see Splinter BonDurant, S. et al. (2002) MagneSil Paramagnetic Particles:
A high-throughput PCR purification system for microarrays. Promega Notes 80, 14–16.
www.promega.com • techserv@promega.com
Quality tested for
success in fluorescent
BigDye® Sequencing
with >98% accuracy
over 600 bases read.
is
m
e
t
sys of
s
i
h
T pable
ca -well
384 ations.
ic
purif
31
PCR Clean-Up
Wizard® PCR Preps
DNA Purification System
Resin/DNA mix
The Wizard PCR Preps DNA Purification System(k)
provides a simple, reliable way to purify double-stranded,
PCR-amplified DNA. PCR products are effectively
separated from contaminants, including primer-dimers
and amplification primers. This system can also be used
to purify DNA fragments from agarose gels. The DNA
can be eluted from the Wizard PCR Preps DNA
Purification Resin in water or TE buffer, with little salt or
macromolecular contamination. A unique feature of this
resin-based method is it’s size cutoff capability. The resin
does not appreciably bind double-stranded DNA smaller
than 200bp, virtually assuring the removal of primerdimers from the reaction. Multiple PCR preps can be
easily processed at one time using the Vac-Man®
Laboratory Vacuum Manifold (Cat.# A7231).
Before Purification
Add resin/DNA
mix to syringe barrel
and apply vacuum.
Add isopropanol,
removing solution
by vacuum.
After Purification
M
M
bp
Transfer
Minicolumn to
microcentrifuge tube.
0935TA11_4A
1,000 –
500 –
200 –
50 –––––
Centrifuge.
Recovery of PCR products using the Wizard PCR Preps DNA Purification
System. Equivalent amounts of a PCR reaction taken before and after purification were
separated on a 1% agarose gel and stained with ethidium bromide.
2
3
4
Transfer Minicolumn
to new tube.
Add water.
5
Centrifuge to elute
DNA.
2819MB04_1A
1
Overview of PCR product purification using the Wizard PCR Preps DNA
Purification System and the Vac-Man Laboratory Vacuum Manifold.
0348TA08_3A
Wizard® PCR Preps DNA Purification System
Purification and analysis of PCR products from low-melting agarose using the
Wizard PCR Preps System. Reactions are shown before (lanes 2 and 4) and after (lanes
3 and 5) purification.
32
protocol
d
e
s
a
b
Syringe
(where
d
e
d
i
v
also pro
quired).
e
r
s
i
m
no vacuu
Cat.#: A7170 (50 preps)
A2180 (250 preps)
Protocol:
www.promega.com/tbs/tb118/tb118.html
Citations for use available at:
www.promega.com/citations/
Promega DNA Analysis Notebook
PCR Clean-Up
PCR Clean-Up Systems and Accessories
Product
Wizard® SV Gel and PCR Clean-Up System(k)
Size
Cat.#
Size
Cat.#
50 preps
250 preps
A9281
A9282
For Laboratory Use. Manual spin-basket system for direct PCR purification and gel isolation from standard agarose gels.
Product
Wizard® SV 96 PCR Clean-Up System*
Vac-Man® 96 Vacuum Manifold
1 × 96 preps
4 × 96 preps
8 × 96 preps
1 each
A9340
A9341
A9342
A2291
* For Laboratory Use. The Wizard® SV 96 PCR Clean-Up System provides a manual or automated system for 96-well direct PCR purification by vacuum. Compatible with a wide variety of PCR
additives and all Promega amplification reaction buffers.
Product
Wizard® MagneSil® PCR Clean-Up System(a)*
Wizard® MagneSil® PCR Clean-Up System, HTP1(a)*
MagnaBot® 96 Magnetic Separation Device
MagnaBot® 384 Magnetic Separation Device
MagnaBot® Spacer (3/16”)
384-Well Plate, Flat
Size
4 × 96 preps
8 × 96 preps
100 × 96 preps
1 each
1 each
1 each
10 pack
Cat.#
A1930
A1931
A1935
V8151
V8241
V8381
V5291
* For Laboratory Use. The Wizard® MagneSil® PCR Clean-Up System provides an automated 96- or 384-well system for direct purification of PCR products. Compatible with standard reactions
using Promega's PCR Master Mix or GoTaq® DNA Polymerase.
Product
Wizard® PCR Preps DNA Purification System(k)*
Vac-Man® Laboratory Vacuum Manifold, 20-sample capacity
Size
50 preps
250 preps
1 each
Cat.#
A7170
A2180
A7231
* For Laboratory Use. The Wizard® PCR Preps DNA Purification System is a manual, resin-based vacuum system for direct purification or isolation from agarose gels.
Related Products
Product
Agarose, LE, Analytical Grade
Agarose, Low Melting Point, Analytical Grade
Blue/Orange Loading Dye, 6X*
TAE Buffer, 10X
TAE Buffer, 40X
TBE Buffer, 10X
Size
100g
500g
25g
3ml (3 × 1ml)
1,000ml
1,000ml
1,000ml
Cat.#
V3121
V3125
V2111
G1881
V4271
V4281
V4251
*For Laboratory Use.
www.promega.com • techserv@promega.com
33
Cloning PCR DNA
Overview
The fact that amplicons generated with Taq DNA
Polymerase typically have A overhangs led to the
method referred to as T-vector cloning. In essence, the
plasmid cloning vector is engineered to contain 3′-T
overhangs that match the 3′-A overhang of the amplicon
(6). The A-tailed amplicon is directly ligated to the Ttailed plasmid vector, and there is no need for further
enzymatic treatment of the amplicon other than the
action of T4 DNA Ligase. Promega has systems based
on this technology for routine subcloning and direct
mammalian expression.
Cloning PCR products into plasmid vectors is a
common downstream application of PCR. When PCR
was in its infancy, researchers found that it was not easy
to clone PCR products by simple blunt-end ligation into
blunt-ended plasmid vectors because some
thermostable DNA polymerases, including Taq DNA
Polymerase, add a single nucleotide base extension to
the 3′-end of blunt DNA in a template-independent
fashion (1,2). Most commonly the base added is
adenine, leaving what is called an “A overhang.” To
overcome this, researchers had to treat PCR products to
blunt-end the PCR fragments prior to cloning. Such
experiments often suffered from low cloning efficiencies.
References
1. Clark, J.M. (1988) Novel non-template nucleotide addition reactions
catalyzed by procaryotic and eucaryotic DNA polymerases. Nucl. Acids Res.
16, 9677–86.
2. Mole, S.E., Iggo, R.D. and Lane, D.P. (1989) Using the polymerase chain
reaction to modify expression plasmids for epitope mapping. Nucl. Acids
Res. 17, 3319.
3. Scharf, S.J., Horn, G.T. and Erlich, H.A. (1986) Direct cloning and sequence
analysis of enzymatically amplified genomic sequences. Science 233,
1076–78.
4. Kaufman, D.L. and Evans, G.A. (1990) Restriction endonuclease cleavage at
the termini of PCR products. BioTechniques 9, 304–6.
5. Digestion of restriction sites close to the end of linear DNA. In: Restriction
Enzyme Resource Guide. Promega Corporation.
www.promega.com/guides/re_guide/chaptwo/2_6.htm
6. Mezei, L.M. and Storts, D.R. (1994) Cloning PCR Products. In: PCR
Technology Current Innovations. Griffin, H.G. and Griffin, A.M. (eds).
CRC Press, pp. 21–7.
Another commonly used strategy for PCR cloning is to
add restriction enzyme recognition sites to the ends of
PCR primers (3). The PCR product is then digested and
cloned into the desired vector. When using this method,
care must be exercised in primer design because not all
enzymes cleave efficiently at the ends of DNA fragments,
and you may not be able to use every enzyme you
desire (4,5). Some enzymes require extra bases outside
the restriction enzyme recognition site, adding further
expense to the PCR primers as well as increasing the
risk of annealing to unrelated sequences in the genome.
For cloning PCR products, the choice is yours
A
A
• G
reat
for
• D
sequ
rop
enci
out
ng!
inser
singl
t w
e B
ith
st Z
I dig
est
T
T
T
Basic
Subcloning
®
pGEM -T Easy Vector
• Great for sequencing!
• Drop out insert with
single Eco R I, Not I
or Bst Z I digest
34
T
Basic
Subcloning
pGEM®-T Vector
T
Direct Mammalian
Expression
pTARGET™ Mammalian
Expression Vector
3779MA07_2A
T
r
promote
V
M
C
•
or
R gene f
o
e
N
•
n
Selectio
8
1
4
G
ith
insert w
t
u
o
p
o
• Dr
I digest
R
o
c
E
single
Promega DNA Analysis Notebook
Cloning PCR DNA
Select recombinants by
pGEM®-T Vector System II
(supplied with High Efficiency JM109 Competent Cells)
Cat.#: A3610 (20 reactions)
Protocol:
www.promega.com/tbs/tm042/tm042.html
Citations for use of the pGEM®-T System online at:
www.promega.com/citations/
Xmn I 1994
Nae I
2692
Sca I
1875
➞
The most basic need in PCR cloning is for simple, general
cloning vectors. The pGEM-T and pGEM-T Easy Vector
Systems(h,i) were designed for just that purpose. The
vectors are based on the pGEM-5Zf(+) Vector(h) backbone.
The pGEM-T and -T Easy Vectors provide convenient T7
and SP6 promoters that serve as sequencing primer
binding sites and also allow in vitro transcription of either
strand of the insert with the appropriate RNA polymerase.
The vectors also have the lacZα coding region, allowing
easy blue/white screening of recombinants. The pGEM-T
and -T Easy Vectors are provided with 2X Rapid Ligation
Buffer, which allows efficient ligation in just 1 hour with the
supplied T4 DNA Ligase. You can either supply your own
favorite E. coli competent cells or purchase the system with
Promega’s high-efficiency JM109 Competent Cells. The
choice is yours.
T7
f1 ori
Amp r
pGEM®-T
Vector
(3000bp)
lacZ
T
T
Spe I
Not I
BstZ I
Pst I
Sal I
Nde I
Sac I
BstX I
Nsi I
ori
blue/white selection.
What is Blue/White Selection?
The enzyme β-galactosidase, the product of the
bacterial lacZ gene, can be separated into two
domains—the α-fragment and the ω-fragment. The two
fragments interact to form a functional β-galactosidase.
For blue/white selection, the ω-fragment is expressed
by the E. coli host strain, and the α-fragment is
provided by the cloning vector. An intact, in-frame
α-fragment will interact with the host strain
ω-fragment, creating functional β-galactosidase. This is
known as α-complementation. Bacteria capable of
producing functional β-galactosidase will cleave the
substrate X-Gal (5-bromo-4-chloro-3-indolyl-β-Dgalactopyranoside), creating blue colonies when grown
on indicator plates containing IPTG and X-Gal (see
recipe on p. 40). Blue/white-capable cloning vectors
have a multiple cloning site within the α-fragment
coding sequence. When your sequence of interest is
inserted within this region, the α-fragment is disrupted,
α-complementation does not occur, and the colony is
white. E. coli (e.g., JM109, DH5α™ or XL1-Blue)
transformed with an insert-containing plasmid produce
white colonies, while those containing empty or
religated vector produce blue colonies.
www.promega.com • techserv@promega.com
Apa I
Aat II
Sph I
BstZ I
Nco I
Sac II
SP6
Drop out your
insert with a
single Bst Z I
digest.
1 start
14
20
26
31
37
46
55
62
62
73
75
82
94
103
112
126
0356VA04_3A
pGEM®-T and pGEM®-T Easy Vector Systems
pGEM®-T Vector System I
(you supply the competent cells)
Cat.#: A3600 (20 reactions)
➞
Basic Subcloning
with:
s
t
r
nse
r
nce i
e
u
q
e
Prime
S
r
e
t
o
r
Prom
SP6
Prime
r
e
t
o
rimer
m
o
P
r
P
d
r
a
T7
rw
r
13 Fo
Prime
M
/
e
s
C
r
U
e
p
ev
13 R
M
/
C
pU
Need sequencing-grade plasmid DNA?
Promega has the system.
Wizard® Plus SV Minipreps DNA Purification System(q,r)
Simple spin preps for plasmid DNA. Guaranteed for
fluorescent sequencing.
Cat.#: A1330 (50 preps)
Cat.#: A1460 (250 preps)
Protocol:
www.promega.com/tbs/tb225/tb225.html
35
Cloning PCR DNA
pGEM®-T Easy Vector System I
(you supply the competent cells)
Cat.#: A1360 (20 reactions)
➞
Xmn I 2009
Sca I 1890
Nae I 2707
f1 ori
pGEM-T Easy
Vector
lacZ
T
T
(3015bp)
pGEM®-T Easy Vector System II
(supplied with High Efficiency JM109 Competent Cells)
Cat.#: A1380 (20 reactions)
64
Spe I
70
EcoR I
77
Not I
77
BstZ I
88
Pst I
90
Sal I
97
Nde I
109
Sac I
Bst X I 118
127
Nsi I
141
SP6
➞
ori
Protocol:
www.promega.com/tbs/tm042/tm042.html
Drop out your
insert with a
single Bst Z I,
Eco R I or
Not I digest.
Citations for use of the pGEM-T Easy System
online at:
www.promega.com/citations/
1473VA05_6A
Amp r
T7
1
start
Apa I
14
Aat II
20
Sph I
26
BstZ I
31
Nco I
37
BstZ I
43
Not I
43
Sac II
49
EcoR I
For maximum subcloning efficiency, purify the PCR
product before subcloning. The presence of PCR
primers and primer-dimers can reduce subcloning
efficiency. See Chapter 3 for more information.
Sequence inserts with:
SP6 Promoter Primer
T7 Promoter Primer
pUC/M13 Forward Primer
pUC/M13 Reverse Primer
If you do not purify your PCR product, at least make
the amplification as specific as possible. The cleaner
the product, the better the ligation efficiency. Try to
avoid production of primer-dimers by optimizing the
amplification reaction conditions. See Chapter 2 for
more information on optimizing PCR.
450
For maximum efficiency,
use competent cells
capable of at least
1 x 108cfu/µg DNA.
400
Number of White Colonies
350
300
250
200
150
Example of Transformation Efficiency Calculation:
100
0
0
2
4
6
8
Hours
10
12
14
16
4019MA03_3A
50
Relationship between incubation time and cloning efficiency using the 2X
Rapid Ligation Buffer and the pGEM-T Easy Vector. The Control Insert DNA
supplied with the pGEM-T Easy Vector was ligated into the vector using a 1:1 vector:insert
molar ratio. The Rapid Ligation Buffer and T4 DNA Ligase were used in ligation reactions,
which were set up at room temperature (24°C) and allowed to proceed from 0.25 to 16
hours. Number of white colonies (transformants) versus time of ligation are shown. This
graph was adapted from Table 2 in Frackman, S. and Kephart, D. (1999) Rapid Ligation for
the pGEM-T and pGEM-T Easy Vector Systems. Promega Notes 71, 8–10.
36
After 100µl competent cells are transformed with
0.1ng uncut plasmid DNA, the transformation
reaction is added to 900µl of SOC medium
(0.1ng DNA/ml). A 1:10 dilution with SOC medium
(0.01ng DNA/ml) is made, and 100µl is plated on
each of two plates (0.001ng DNA/100µl). If 200
colonies are obtained (average of two plates), what is
the transformation efficiency?
200cfu × 1ng = 2 × 108 cfu/µg DNA
0.001ng 10–3µg
Promega DNA Analysis Notebook
Cloning PCR DNA
pTARGET™ Mammalian Expression Vector System
The pTARGET Mammalian Expression Vector(i,j) is
designed to streamline your experiments, allowing you
to go from PCR and T-vector cloning directly to
expression analysis in a mammalian system. The
pTARGET Vector is based on the popular pCI-neo
Vector(h,j) (Cat.# E1841) and delivers powerful
mammalian expression through the CMV promoter. The
vector also has the neomycin resistance necessary for
G-418 Sulfate selection of stable transformants.
pTARGET™ Mammalian Expression Vector System
Cat.#: A1410 (20 reactions and 20 transformations
with high-efficiency JM109 Competent Cells)
Protocol:
www.promega.com/tbs/tm044/tm044.html
Citations for use of the pTARGET System online at:
www.promega.com/citations/
ammalian
m
y
l
n
o
The
r
T-vecto
n
o
i
s
s
e
r
exp
e/white
u
l
b
f
o
capable
g.
screenin
The pTARGET Vector is the only mammalian expression
T-vector offering blue/white selection of recombinants.
The vector contains the lacZ α-peptide fragment to
complement the ω-fragment of lacZ that is expressed in
common E. coli strains like JM109, DH5α™ and XL-1
Blue. See page 35 for more explanation of blue/white
selection.
Sgf I 664
ori
lacZ
CMV
Enhancer/Promoter
I-Ppo I
851
Intron
Ampr
T
pTARGET™
Vector
(5670bp)
Synthetic
poly(A)
Neo
T
SV40 Late
poly (A)
fl ori
SV40 Enhancer/
EarlyPromoter
➞
:
erts with
s
in
e
c
n
e
Sequ
r
ter Prime
T7 Promo
pTARGET
g Primer
Sequencin
Bgl II 5665
T7
EcoR I
BamH I
Nhe I
Xho I
Mlu I
1250
1256
1264
1270
1276
T overhangs
Sma I
Kpn I
Sal I
Acc I
Not I
EcoR I
1293
1301
1303
1304
1311
1318
lacZ
pTARGET Mammalian Expression Vector has been used
for transient expression in many cell lines including:
COS-1 SV40-transformed monkey kidney
COS-7 SV40-transformed monkey kidney
H9c2 rat myoblast
McA-RH7777 rat hepatoma
Primary human melanoma
The pTARGET Mammalian Expression Vector has been
used to create stable transfectants by G-418 Sulfate
selection in many cell lines including:
1376 TCC human bladder transitional cell carcinoma
293 human embryonic kidney cell
A549 human adenocarcinoma
CHO Chinese hamster ovary
NIH/3T3 mouse fibroblast
J82 human bladder transitional cell carcinoma
PS120 Chinese hamster lung fibroblast
RAW264.7 mouse monocyte/macrophage cell line
T24 human bladder transitional cell carcinoma
U937 human leukemic cells
See Promega’s online citation database for further
examples and details: www.promega.com/citations/
www.promega.com • techserv@promega.com
1505VA07_6A
Direct Mammalian Expression
Drop out your insert
with a single
Eco R I digest.
The pTARGET Vector contains the simian virus 40
(SV40) enhancer and early promoter region
upstream of the neomycin phosphotransferase gene.
The SV40 early promoter contains the SV40 origin of
replication, which will induce transient, episomal
replication of the pTARGET Vector in cells expressing
the SV40 large T antigen such as COS-1 or COS-7
cells (1). Consequently, the copy number of the
vector will increase in these SV40-transformed cell
lines and give higher transient expression than in
other cell types.
1. Gluzman, Y. (1981) SV40-transformed simian cells support the
replication of early SV40 mutants. Cell 23, 175–82.
37
Cloning PCR DNA
Direct Mammalian Expression
(continued)
pTARGET™ Mammalian Expression Vector System
7
6
pCI-neo
pTARGET ™
5
4
3
2
1
0
HeLa
NIH3T3
140
120
100
80
60
40
20
0
Cells Transfected
D.
40
35
30
25
pCI-neo
pTARGET ™
20
15
10
5
0
HeLa
NIH3T3
HeLa
Cells Transfected
NIH3T3
Cells Transfected
E.
2.5
2
1.5
1
0.5
0
HeLa
60
pCI-neo
50
pTARGET ™
Experimental details
available at:
www.promega.com/pnotes/
58/5189a/5189a.html
40
30
20
10
NIH3T3
0
Cells Transfected
HeLa
NIH3T3
Cells Transfected
1539MA07_6A
3
pCI-neo
pTARGET ™
β-galactosidase Activity
(units/plate × 10-3)
3.5
Firefly Luciferase Activity
(T.U./plate × 106)
pCI-neo
pTARGET ™
CAT Activity
(CPM/plate × 106)
8
Human Growth Hormone
Activity (ng/ml cond. medium)
Renilla Luciferase Activity
(T.U./plate × 106)
C.
B.
A.
Expression of various reporter proteins using either the pTARGET Mammalian Expression Vector or the pCI-neo Mammalian Expression Vector. The pTARGET Vector was
designed from the pCI-neo Vector (Cat.# E1841). Vector sequences for blue/white selection do not interfere with expression. T.U. = Turner light units. Details of this experiment may be found
in Brondyk, B. (1996) pTARGET Vector: A new mammalian expression T-Vector. Promega Notes 58, 2–7.
For expression in mammalian systems, your amplicon
should contain an initiation AUG codon and a stop
codon. Ideally the AUG codon is in the context of a
Kozak consensus sequence: (A or G)CCAUGG (1).
Be sure the initiation codon is the first AUG codon
encountered in the sequence.
1. Kozak, M. (1987) At least six nucleotides preceding the AUG initiator
codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947–50.
Learn about transfection and tools available from
Promega in the Transfection Guide available online at:
www.promega.com/guides/, or request literature
#BR041 from your local Promega representative.
Need transfection grade plasmid DNA?
Promega has the system.
Wizard MagneSil Tfx™ System(s)
For automated 96-well transfection-grade plasmid
DNA purification.
Cat.#: A2380 (4 × 96 preps)
A2381 (8 × 96 preps)
Protocol:
www.promega.com/tbs/tb314/tb314.html
4432CA
For information on automated methods visit:
www.promega.com/automethods/
38
Promega DNA Analysis Notebook
Cloning PCR DNA
PCR Cloning Techniques
Giving Blunt-Ended DNA an A-Tail for T-Vector
Subcloning
Rapid PCR-Based Screen for Orientation of Insert
PCR amplicons generated with proofreading
polymerases like Pfu or Tli DNA Polymerase are bluntended. Promega has developed an easy method to add
an A-tail to PCR products generated using these
polymerases so that they will become suitable
substrates for T-vector cloning. Full details of the
protocol are available in the pGEM-T and pGEM-T Easy
Vector Systems Technical Manual #TM042.
All Promega PCR cloning vectors have some unique
landmarks, including RNA promoter primer binding sites
allowing easy sequencing of inserts. The primer binding
sites can also be used to rapidly screen for insert
orientation. For example, we cloned a 1.8kb insert into
the pGEM®-T Easy Vector System (1). The insert could
be oriented in one of two ways—toward the T7
promoter or toward the SP6 promoter:
T7 Orientation
T7 Primer
Start with 1–7µl of purified PCR fragment generated by a
proofreading polymerase (e.g., Pfu DNA Polymerase).
Forward Primer
Add 1µl Taq DNA Polymerase 10X Reaction Buffer with MgCl2.
1.8kb fragment
1.8kb fragment
T7
Add dATP to a final concentration of 0.2mM.
Reverse Primer SP6 Primer
SP6
Forward Primer
SP6 Primer
To check for orientation, we performed colony PCR with
the T7 Promoter Primer and either the gene-specific
forward PCR primer or reverse PCR primer. Eight white
colonies with inserts were chosen from the cloning
experiment for orientation analysis. Clones with the T7
orientation will produce the fragment with only the
reverse PCR primer, and clones in the opposite (SP6)
orientation will only produce a product with the forward
PCR primer as illustrated below.
T7 + For.
T7 + Rev.
M 1 2 3 4
1 2 3 4
bp
2,645–
1,605–
1,198–
Add 5 units of Taq DNA Polymerase.
Add deionized water to a final reaction volume of 10µl.
Incubate at 70°C for 15–30 minutes.
Use 1–2µl in a ligation reaction with
Promega’s pGEM®-T and pGEM®-T Easy Vector.
2357MA02_9A
SP6 Orientation
T7 Primer Reverse Primer
SP6
2574MB02_9A
T7
An A-tailing procedure for blunt-ended PCR fragments.
Ends Left by Various Thermostable Polymerases.
Taq DNA Polymerase
3′A overhang*
GoTaq® DNA Polymerase
3′A overhang*
Tfl DNA Polymerase
3′A overhang*
Tth DNA Polymerase
3′A overhang*
Pfu DNA Polymerase
Blunt
Tli DNA Polymerase
Blunt
Long PCR mixes
Blunt
Proofreading Polymerases
Blunt
* All bases may be found at 3′ overhang. A tends to occur most often.
222–
2575TA02_9A
676–
517–
Colony PCR. Colonies were suspended in 50µl sterile water, boiled for 10 minutes,
centrifuged at 16,000 × g for 5 minutes, and 5µl of the supernatant was used in each
amplification. The DNA was amplified by PCR in 50µl volumes with 50pmol of each primer
and 1.25 units of Promega’s Taq DNA Polymerase (Cat.# M1661). After an initial
denaturation of 2 minutes at 94°C, the amplification profile was 35 cycles of denaturation
(94°C for 30 seconds), annealing (55°C for 1 minute) and extension (72°C for 2.5
minutes); PCR was concluded with 1 cycle of 72°C for 10 minutes. Amplification products
(8µl) were analyzed on a 1% agarose gel containing ethidium bromide.
Reference
For more information and techniques
for cloning PCR DNA, check out
Promega's Frequently Asked Questions
on the T-vector cloning systems at:
www.promega.com/faq/
1. Knoche, K. and Kephart, D. (1999) Cloning blunt-end Pfu DNA polymerasegenerated PCR fragments into pGEM-T Vector Systems. Promega Notes 71,
10–13.
www.promega.com • techserv@promega.com
39
Cloning PCR DNA
PCR Cloning Techniques
(continued)
What PCR Cloning Controls Can Do For You
Each Promega PCR cloning system is provided with a
Control Insert. The ligation and subsequent transformation
of the Control Insert can give you a lot of information
about your ligation and transformation reactions.
The total number of blue colonies in Control Insert and
no-insert controls should be approximately equal. The
negative control may have some white colonies.
Typical PCR Cloning Results Using pGEM®-T
Easy Vector and JM109 Competent Cells
(1.5 × 108 cfu/ng DNA).
Efficiency
% White
(cfu/ng DNA)
Colonies
Control Insert
1,110
92%
Control Insert
1,125
92%
No insert
92
0%
No insert
109
0%
Ligations performed at room temperature for 1 hour.
Bacterial Plates for Blue/White Selection
LB medium (per liter)
10g Bacto®-tryptone
5g Bacto®-yeast extract
5g NaCl
Adjust pH to 7.0 with NaOH.
Ampicillin Stock Solution
Dissolve at 50mg/ml in water. Filter sterilize. Store in
aliquots at –20°C
IPTG stock solution (0.1M)
1.2g IPTG (Cat.# V3951)
Add water to 50ml final volume. Filter-sterilize and
store at 4°C.
X-Gal (2ml)
100mg X-Gal (Cat.# V3941)
Dissolve in 2ml N,N′-dimethyl-formamide. Cover with
aluminum foil and store at –20°C.
Interpreting Results
Results with the experimental insert look like those
with the Control Insert in terms of efficiency and %
white colonies.
Successful experiment. Greater than 80% of the white
colonies should contain inserts.
Results with the experimental insert and Control
Insert look like the negative control.
Ligation has failed. Avoid multiple freeze/thaws of the
ligation buffer. Ligase buffer contains ATP and could
be damaged by freeze-thaws. You may need to aliquot
the ligase buffer into useful portions for your
experimental needs.
Few/No colonies with experimental insert, Control
Insert or negative control.
Transformation has failed. Reassess the competent
cells with an intact, supercoiled plasmid and
determine the transformation efficiency. Use cells
>1 × 108cfu/µg to insure >100 colonies from the
Control Insert ligation.
Experimental insert gives more blue colonies than
the Control Insert or negative control and less white
colonies than the Control Insert.
In-frame insertion with no interruption of the
α-fragment. Although the pGEM-T Vector Control
Insert will produce recombinants that generate white
colonies, the insertion of other DNA fragments into the
lacZ coding sequence may not result in white colonies
unless the fragments disrupt the lacZ reading frame.
Although this tends to occur most frequently with PCR
products of 500bp or less, inserts of up to 2kb have
been reported to result in blue colonies. Moreover,
some insert DNAs can also give pale blue colonies or
“bull’s eye” colonies that have a blue center and a
white perimeter. In one case, we found that a 1.8kb
insert produced white colonies when oriented in one
direction and bull’s eye colonies when oriented in the
opposite direction (1).
1. Knoche, K. and Kephart, D. (1999) Cloning blunt-end Pfu DNA
polymerase-generated PCR fragments into pGEM-T Vector Systems.
Promega Notes 71, 10–13.
LB plates with ampicillin/IPTG/X-Gal
Add 15g agar to 1 liter of LB medium. Autoclave.
Allow the medium to cool to 50°C before adding
ampicillin to a final concentration of 100µg/ml, then
supplement with 0.5mM IPTG and 80µg/ml X-Gal and
pour the plates. Pour 30–35ml of medium into 85mm
petri dishes. Let the agar harden. Store at 4°C for up
to 1 month or at room temperature for up to 1 week.
40
Promega DNA Analysis Notebook
Cloning PCR DNA
Basic Subcloning
Product
pGEM®-T Vector System I(h,i)
pGEM®-T Vector System II(h,i)
pGEM®-T Easy Vector System I(h,i)
pGEM®-T Easy Vector System II(h,i)
Size
Cat.#
Size
Cat.#
Size
Cat.#
Size
Cat.#
20 reactions
20 reactions
20 reactions
20 reactions
A3600
A3610
A1360
A1380
For Laboratory Use. pGEM®-T and pGEM®-T Easy Vector Systems I do not include competent cells. With System II, competent cells are provided.
Direct Mammalian Expression
Product
pTARGET™ Mammalian Expression Vector System(i,j)
20 reactions
A1410
Competent Cells provided.
Primers
Product
T7 Promoter Primer (10µg/ml)[5′-d(TAATACGACTCACTATAGGG)-3′]
SP6 Promoter Primer (10µg/ml) [5′-d(TATTTAGGTGACACTATAG)-3′]
pUC/M13 Forward Primer (10µg/ml) [5′-d(CGCCAGGGTTTTCCCAGTCACGAC)-3′]
pUC/M13 Reverse Primer (10µg/ml) [5′-d(TCACACAGGAAACAGCTATGAC)-3′]
pTARGET™ Sequencing Primer (10µg/ml) [5′-d(TTACGCCAAGTTATTTAGGTGACA)-3′]
PinPoint™ Vector Sequencing Primer [5′-d(CGTGACGCGGTGCAGGGCG)-3′]
2µg
2µg
2µg
2µg
2µg
2µg
Q5021
Q5011
Q5601
Q5421
Q4461
V4211
DNA Purification Systems
Product
Wizard® Plus SV Minipreps DNA Purification System(q,r)*
Wizard MagneSil Tfx™ System(s)
(Automated transfection-grade plasmid purification.)
*For Laboratory Use.
www.promega.com • techserv@promega.com
50 preps
250 preps
4 × 96 preps
8 × 96 preps
A1330
A1460
A2380
A2381
41
Cloning PCR DNA
Accessory Products
Product
Size
Cat.#
Size
Cat.#
Select96™ Competent Cells (Single aliquot competent cells, use 1 to 96 at a time, >1 × 108 cfu/µg DNA)
1 × 96 preps
JM109 High Efficiency Competent Cells (108cfu/µg)* (Packaged 5 × 200µl; use 50µl per transformation >1 × 108 cfu/µg DNA)
1ml
IPTG, Dioxane-Free
5g
50g
X-Gal (50mg/ml)
100mg
Antibiotic G-418 Sulfate (potency >500µg/mg)
100mg
1g
5g
Antibiotic G-418 Sulfate Solution (potency 40–60mg/ml)
20ml
L3300
L2001
V3951
V3953
V3941
V7981
V7982
V7983
V8091
*For Laboratory Use.
Transfection Reagents
Product
TransFast™ Transfection Reagent(t)
Tfx™-10 Reagent(u)
Tfx™-20 Reagent(u)
Tfx™-50 Reagent(u)
42
1.2mg
9.3mg
4.8mg
2.1mg
E2431
E2381
E2391
E1811
Promega DNA Analysis Notebook
DNA Analysis Tools
Benchtop DNA Markers
Ready-to-load markers premixed
with Promega’s Blue/Orange
Loading Dye. The markers are
stable at room temperature so
you can store them on your
bench top!
Read
y-toload
mark
ers y
ou sto
on yo
re
ur be
nch t
op
bp
bp
– 2,645
bp
– 1,605
– 1,353
– 1,198
– 1,078
– 10,000
–––––––– 8,000
– 6,000
– 5,000
– 4,000
bp
– 872
– 676
– 603
– 1,000
– 750
– 517
– 460
– 396
– 350
– 310
– 281/271
– 234
– 194
– 222
– 179
– 500
– 3,000
– 2,500
– 2,000
– 300
– 1,500
– 150
– 50
– 1,000
– 750
– 118
– 126
– 500
– 72
– 75/65
[51,36]
– 250,253
2% agarose
2% agarose
2% agarose
BenchTop φX174
DNA/Hae III Markers
Cat.# G7511
Load 5µl/lane
BenchTop pGEM®
DNA Markers
Cat.# G7521
Load 5µl/lane
BenchTop PCR Markers
Cat.# G7531
Load 6µl/lane
0.7% agarose
BenchTop 1kb DNA Ladder
Cat.# G7541
Load 6µl/lane
Ladders and Digest Markers
Promega’s ladders and digest markers allow you to
choose the markers you want, mix and load as much as
you wish. Each marker comes with a tube of Blue/Orange
Loading Dye, 6X for both the marker and your samples.
bp
bp
– 1,353
– 1,500
– 1,000
– 900
– 800
– 700
bp
– 800
––––– 750
– 700
––––– 650
– 600
––––– 550
– 500
– 450
– 400
– 350
– 300
– 250
– 200
– 400
– 150
– 10,000
–––––––– 8,000
– 6,000
– 5,000
– 4,000
– 300
– 3,000
– 2,500
– 2,000
– 200
– 1,500
– 1,000
– 100
– 603
– 23,130
– 9,416
– 500
– 6,557
– 4,361
– 310
––––––
281 271
– 2,322
– 2,027
– 234
[564, 125]
– 194
– 750
– 50
– 500
– 250,253
1258TB09_5A
0973TC03_5A
2053TA02_8A
– 100
2054TA02_8A
bp
2% agarose/TAE
2% agarose/TAE
2% agarose
25bp DNA Step Ladder
Cat.# G4511
Load 5µl/lane
50bp DNA Step Ladder
Cat.# G4521
Load 5µl/lane
100bp DNA Ladder
Cat.# G2101
Load 5µl/lane
www.promega.com • techserv@promega.com
0.7% agarose
1kb DNA Ladder
Cat.# G5711
Load 5µl/lane
0.7% agarose
Lambda DNA/
Hind III Markers
Cat.# G1711
Load 1µl/lane
1238TA09_5A
– 25
bp
– 600
bp
– 300
––––– 275
– 250
––––– 225
– 200
– 175
– 150
– 125
– 100
– 75
– 50
– 1,078
– 872
– 118
[72]
8% acrylamide
φX174 DNA/Hae III Markers
Cat.# G1761
Load 1µl/lane
43
U.S. Pat. Nos. 6,027,945 and 6,368,800, Australian Pat. No. 732756 and other patents and patents pending.
This product may be covered by one or more of the following patents issued to Promega Corporation for multiplex amplification of STR loci: U.S. Pat. Nos. 5,843,660, 6,479,235 and
6,221,598 and Australian Pat. No. 724531. Other patents are pending.
(c) U.S. Pat. No. 6,238,863 and other patents pending.
(d) STR loci are the subject of U.S. Pat. No. 5,766,847, German Pat. No. DE 38 34 636 C2 and other patents issued to the Max-Planck-Gesellschaft zur Förderung der Wissenschaften, eV,
Germany. The development and use of STR loci are covered by U.S. Pat. No. 5,364,759, Australian Pat. No. 670231 and other pending patents assigned to Baylor College of Medicine,
Houston, Texas.
Use of Promega's STR Systems requires performance of the polymerase chain reaction (PCR), which is the subject of European Pat. Nos. 201,184 and 200,362 and U.S. Pat. Nos. 4,683,195,
4,965,188 and 4,683,202 owned by Hoffmann-La Roche. Purchase of Promega's STR Systems does not include or provide a license with respect to these patents or any other PCR-related
patent owned by Hoffmann-La Roche or others. Users of Promega's STR Systems may, therefore, be required to obtain a patent license, depending on the country in which the system is
used. For more specific information on obtaining a PCR license, please contact Hoffmann-La Roche.
(e) Certain applications of this product are covered by patents issued and applicable in certain countries. Because purchase of this product does not include a license to perform any patented
application, users of this product may be required to obtain a patent license depending upon the particular application and country in which the product is used.
(f) The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of
this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license.
(g) U.S. Pat. No. 6,242,235 and other patents pending.
(h) U.S. Pat. No. 4,766,072.
(i) Licensed under one or more of U.S. Pat. Nos. 5,487,993 and 5,827,657 and European Pat. No. 0 550 693.
(j) The CMV promoter and its use are covered under U.S. Pat. Nos. 5,168,062 and 5,385,839 owned by the University of Iowa Research Foundation, Iowa City, Iowa, and licensed FOR
RESEARCH USE ONLY. Commercial users must obtain a license to these patents directly from the University of Iowa Research Foundation
(k) Licensed under U.S. Pat. No. 5,075,430.
(l) U.S. Pat. No. 5,552,302, Australian Pat. No. 646803 and other patents.
(m) U.S. Pat. Nos. 4,966,964, 5,019,556 and 5,266,687, Australian Pat. Nos. 616881 and 641261 and other pending and issued patents, which claim vectors encoding a portion of human
placental ribonuclease inhibitor, are exclusively licensed to Promega Corporation.
(n) U.S. Pat. Nos. 5,324,637, 5,492,817 and 5,665,563, Australian Pat. No. 660329 and other patents.
(o) The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673.
(p) The use of dUTP in combination with UNG is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of
this process. Use of this product is recommended for persons that either have a license to perform this procedure or are not required to obtain a license.
(q) U.S. Pat. No. 5,981,235 and Australian Pat. No. 729932 have been issued to Promega Corporation for methods for isolating nucleic acids using alkaline protease. Other patents are pending.
(r) Australian Pat. No. 730718 and Singapore Pat. No. 64532 have been issued to Promega Corporation for an improved filtration system and method. Other patents are pending.
(s) U.S. Pat. No. 6,194,562, Australian Pat. No. 740145 and other patents pending.
(t) The cationic lipid component of the TransFast™ Transfection Reagent is covered by U.S. Pat. Nos. 5,824,812, 5,869,715 and 5,925,623, Australian Pat. No. 713093 and pending foreign
patents.
(u) The cationic lipid component of the Tfx™ Reagents is covered by U.S. Pat. Nos. 5,527,928, 5,744,625 and 5,892,071, Australian Pat. No. 704189 and other pending foreign patents.
(a)
(b)
GoTaq, MagnaBot, MagneSil, pGEM, TNT, Vac-Man and Wizard are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office. ImProm-II, PinPoint,
pTARGET, Select96, SoftLink, TaqBead, TetraLink, Tfx and TransFast are trademarks of Promega Corporation.
ABIPRISM and Genotyper are registered trademarks of Applera Corporation. AmpliTaq and AmpliTaq Gold are registered trademarks of Roche Molecular Systems, Inc. Bacto is a registered
trademark of Difco Laboratories, Detroit, Michigan. BigDye is a registered trademark of Applera Corporation. Biomek is a registered trademark of Beckman Instruments, Inc. Cy is a registered
trademark of Amersham Biosciences Ltd. DH5α is a trademark of Life Technologies, Inc. Eppendorf is a registered trademark of Eppendorf-Netheler-Hinz GmbH. Ficoll is a registered trademark
of Amersham Biosciences Ltd. Finnpipette is a registered trademark of Labsystems Oy. MultiPROBE is a registered trademark of the Perkin Elmer Corporation. Oxford Benchmate is a registered
trademark of Sherwood Medical Company. Pipetman is a registered trademark of Rainin Instrument Co. Platinum is a registered tradeark of Invitrogen Corporation. Triton is a registered
trademark of Union Carbide Chemicals & Plastics Technology Corporation. Tween is a registered trademark of ICI Americas, Inc. Vent is a registered trademark of New England Biolabs, Inc.
44
Promega DNA Analysis Notebook
Promega
Corporate
Headquarters
Branch Office
Subsidiary Office
Representative Office
4089MA04_3A
Distributor
Joint Venture
Promega Corporation
2800 Woods Hollow Road
Madison, WI 53711-5399 USA
Tel:
Fax:
Toll-Free:
Toll-Free Fax:
Internet:
608-274-4330
608-277-2516
800-356-9526
800-356-1970
www.promega.com
Promega Biosciences, Inc.
A Division of Promega
Corporation
San Luis Obispo, California
Australia, Sydney
Tel:
02 9565 1100
Fax:
02 9550 4454
Freecall:
1800 225 123
Freefax:
1800 626 017
E-mail:
aus_custserv@au.promega.com
China, Beijing
Tel:
10 6849 8287
Fax:
10 6849 8390
E-mail: promega@promega.com.cn
France, Lyon
Tel:
04 37 22 50 00
Fax:
04 37 22 50 10
Numero Vert:
0 800 48 79 99
E-mail: fr_custserv@fr.promega.com
Germany/Austria, Mannheim
Tel:
(+49) (0) 621-8501-0
Fax:
(+49) (0) 621-8501-222
Free Phone:
00800-77663422
Free Fax
00800-77663423
E-mail:
de_custserv@de.promega.com
Italy, Milan
Tel:
02 54 05 01 94
Fax:
02 55 18 56 64
Numero Verde:
800 69 18 18
E-mail: it_custserv@it.promega.com
Japan, Tokyo
Tel:
03-3669-7981
Fax:
03-3669-7982
E-mail: prometec@jp.promega.com
Belgium/Luxembourg/
The Netherlands, Leiden
Tel:
(+31) (0) 71-5324244
Fax:
(+31) (0) 71-5324907
Free Tel BE:
0800-18098
Free Fax BE:
0800-16971
Free Tel NL:
0800-0221910
Free Fax NL:
0800-0226545
E-mail:
bnl_custserv@nl.promega.com
Pacific Asia Region, Singapore
Tel:
65 6254 5265
Fax:
65 6254 8645
E-mail: nicholas.ng@promega.com
Switzerland, Wallisellen
Tel:
01 878 90 00
Fax:
01 878 90 10
Technical Service:
01 878 90 20
E-mail:
catalys_custserv@promega.com
United Kingdom, Southampton
Tel:
023 8076 0225
Fax:
023 8076 7014
Free Phone:
0800 378994
Free Fax:
0800 181037
E-mail: ukcustserve@promega.com
C
ER
TIFIE
Promega Corporation • 2800 Woods Hollow Road • Madison, WI 53711-5399 USA • Telephone 608-274-4330 • Fax 608-277-2601
LI
T
EM
QU
A
D
TY S
YS
©2004 Promega Corporation. All Rights Reserved.
Prices and specifications subject to change
without prior notice.
Printed in USA Revise 1/04
11250-BR-PU
Part #BR129 Update