CYAN MAGENTA YELLOW BLACK
DIE MARK
TOPO® Cloning Technology
Fast, effective cloning of PCR products
Direct your cloning future.
TOPO® Cloning Technology
With TOPO® Cloning
Technology You Can:
• Clone Taq-amplified,
blunt-end, and long
PCR products
• Sequence or clone
directly into an
expression vector
• Ligate in 5 minutes at
room temperature and
obtain up to 99%
recombinants
These products may be covered by one or more Limited Use Label Licenses (See the Invitrogen catalog or our web site,
www.invitrogen.com). By the use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses.
For research use only. Not intended for any animal or human therapeutic or diagnostic use. Printed in the U.S.A. ©2003 Invitrogen
Corporation. All rights reserved. Reproduction forbidden without permission.
Corporate headquarters:
1600 Faraday Avenue • Carlsbad, CA 92008 USA • Tel: 760 603 7200 • Fax: 760 602 6500 • Toll Free Tel: 800 955 6288 • E-mail: tech_service@invitrogen.com • www.invitrogen.com
European headquarters:
Invitrogen Ltd 3 • Inchinnan Business Park • Fountain Drive • Paisley PA4 9RF, UK • Tel: +44 (0) 141 814 6100 • Fax: +44 (0) 141 814 6260 • E-mail: eurotech@invitrogen.com
710-021849 070803
10M
• Obtain up to 99% recombinants
• Ligate in 5 minutes at room temperature
• Clone Taq-amplified, blunt-end, and long PCR products
With TOPO® Cloning Kits You Can:
TOPO® Cloning Kits
Powerful PCR Cloning Tools
For optimal cloning results, you need a technology that you
can rely on. TOPO® Cloning is the most effective technology
available for cloning PCR products and other DNA molecules.
It yields up to 99% recombinants via a simple 5-minute,
bench-top ligation.
Topoisomerase greatly improves cloning
TOPO® Cloning makes ligation faster and more successful.
3´ thymidine. It cleaves one DNA strand, enabling the
It enables 5-minute, bench-top ligation and yields up to
DNA to unwind. The enzyme then religates the ends of
99% recombinants. This speed and efficiency will save
the cleaved strand and releases itself from the DNA.
you hours of time over other methods of cloning.
To harness the religating activity of topoisomerase, TOPO®
The technology behind TOPO® Cloning
vectors are provided linearized with topoisomerase I
The key to TOPO® Cloning is the enzyme, DNA topoiso-
covalently bound to each 3´ phosphate. This enables the
merase I, which functions both as a restriction enzyme and
vectors to readily ligate DNA sequences with compatible
as a ligase. Its biological role is to cleave and rejoin DNA
ends (Figures 1, 2, and 3) (1). In only 5 minutes at room
during replication. Vaccinia virus topoisomerase I specifi-
temperature, the ligation is complete and ready for
cally recognizes the pentameric sequence 5´-(C/T)CCTT-3´
transformation into E. coli.
and forms a covalent bond with the phosphate group of the
Figure 1 - TOPO TA Cloning® of Taq-amplified DNA
Topoisomerase I recognition sites
5 minutes at
room temperature
AGGG
TTCCC
TOPO
P
P
3´ phosphate
CCCTT
GGGA
+
A
PCR Product
TOPO
A
CCCT T
GGGA A
TOPO
PCR Product
A AGGG
T TCCC
Topoisomerase I
is released
TOPO
TOPO TA Cloning® vector
Taq-amplified PCR product
Ligation complete
Figure 2 - Zero Blunt TOPO Cloning of blunt-end DNA
®
®
Topoisomerase I recognition sites
5 minutes at
room temperature
AAGGG
TTCCC
TOPO
P
P
CCCTT
GGGAA
3´ phosphate
+
TOPO
PCR Product
CCCTT
GGGAA
TOPO
PCR Product
AAGGG
TTCCC
Topoisomerase I
is released
TOPO
Zero Blunt® TOPO® vector
Blunt-end PCR product
Ligation complete
Figure 3 – Directional TOPO Cloning of blunt-end DNA
®
Topoisomerase I recognition sites
P
5 minutes at
room temperature
AAGGG
TTCCC
TOPO
P
CCCTT
GGGAAGTGG
3´ phosphate
+
CACC
GTGG PCR Product
TOPO
Directional TOPO® Cloning Vector
TOPO
CCCTT CACC
GGGAA GTGG PCR Product
TOPO
GTGG
AAGGG
TTCCC
Topoisomerase I
is released
extra GTGG released
Blunt-end PCR Product
(designed with CACC at the
5´ end, no modification
at the 3´ end)
Ligation complete
~1~
www.invitrogen.com
TOPO® Cloning Kits
Three simple steps
TOPO® Cloning requires just three easy steps. Simply combine
TOPO® Cloning means:
your PCR product and a TOPO® Cloning vector in the provided
• Utilizing standard PCR primers
solution, wait five minutes, then transform E. coli (Figure 4).
• Cloning efficiently without ligase or overnight incubations
With TOPO® Cloning, the additional time, steps, and reagents
• Using PCR templates without post-PCR modification or
required for ligase-mediated cloning are eliminated. Table 1
gel purification*
provides conservative estimates of the time saved using
TOPO® Cloning versus other methods.
Figure 4 - The TOPO® Cloning protocol
1. Add 1 µl of PCR reaction to 1 µl of
TOPO® Cloning vector.
Perform PCR with Taq or a
proofreading polymerase.
A
CACC
PCR Product
A
OR
2. Incubate 5 minutes at
room temperature.
3. Transform the competent
E. coli provided.
PCR Product
PCR Product
TOPO
TOPO
55
TOPO®
Cloning
Vector
0
5
10
50
45
15
20
40
35
30
25
*The TOPO® XL PCR Cloning Kit requires a 15-minute gel purification step.
Table 1 – TOPO® TA Cloning vs. other methods of cloning
Steps
TOPO TA Cloning®
TA/UA Cloning
Restriction Enzyme
Cutback and Ligation
Order or prepare
PCR Primers
Special primers containing
extra bases are not required
Special primers containing
extra bases are not required
Add 10 extra bases to each 5´
and 3´ PCR primer to create
restriction sites (6 for the
restriction site, 4 for the
spacing)
Prepare the vector
and PCR product
for ligation
Linearized TOPO® Cloning Vectors
are ready for direct ligation of
unmodified, unpurified PCR
products
TA/UA Cloning vectors are
ready for direct ligation of
unmodified, unpurified PCR
products
1) Digest vector and PCR
product with restriction
enzyme(s)
2) Gel purify the digested
PCR product using
low-melt agarose
Obtain ligation reagents
All required cloning reagents
are included
All required cloning reagents
are included
Purchase ligase, ATP, and
ligation buffer
Prepare or purchase
competent cells
TOPO® Cloning Kits include
One Shot® Competent E. coli
1) With competent cells=0 hrs
2) Without competent cells =
Up to 6 hours
1) Purchase: 0 hours
2) Prepare: 6 hours
Incubate the ligation
5 minutes
1 hour
2 to 23 hours
Recombination efficiency up to 99%
60% to 80%
~ 60%
Time required for
cloning
1 to 12 hours
2 to 23 hours
Toll Free: 800 955 6288
5 minutes
~2~
TOPO® Cloning Kits
Complete product offering
Whether you’re PCR cloning with Taq DNA polymerase or a proofreading enzyme, there is a TOPO® Cloning Kit
available to take you quickly and efficiently to the next step. Use Tables 2 and 3 to find the right products for your
specific application. For a complete list of products, please visit our web site at www.invitrogen.com/topo.
Table 2 – TOPO® Cloning product guide
Amplification Enzyme
Application of Cloned PCR product
Taq DNA polymerase:
Product
Page no.
TOPO TA Cloning® Kit
General subcloning
Cloning®
4
Platinum® Taq
in vitro transcription
TOPO TA
DNA Polymerase†
Sequencing
TOPO TA Cloning® for Sequencing
7
High-throughput studies
HTP TOPO TA Cloning® Kit
HTP TOPO TA Cloning® Kit Dual Promoter
HTP TOPO TA Cloning® for Sequencing
13
13
13
Non-directional expression in E. coli, yeast,
insect, or mammalian cells
Various non-directional TOPO® Expression Vectors
8
Zero Blunt® TOPO® Cloning Kit
5
Zero Blunt® TOPO® Cloning Kit for Sequencing
7
Proofreading polymerase:
General subcloning
Platinum® Pfx
in vitro transcription
DNA Polymerase†
Sequencing
Blunt®
Kit Dual Promoter
TOPO®
High-throughput studies
HTP Zero
PCR Cloning Kit
for Sequencing
HTP Directional TOPO® pENTR™ vectors
13
Proofreading polymerase:
Directional expression in E. coli
Champion™ pET Directional TOPO® Expression Kit
10
Platinum® Pfx
DNA Polymerase†*
Directional expression in mammalian cells
pcDNA™3.1 Directional TOPO® Expression Kit
pcDNA™4/HisMax TOPO® TA Expression Kit
ViraPower™ Lentiviral Directional TOPO®
Expression System
11
Expression of cloned PCR products in
multiple hosts (via Gateway® System)
Directional TOPO® pENTR™ vectors
pcDNA/GW/D-TOPO®
12
General subcloning
in vitro transcription
Sequencing
TOPO® XL PCR Cloning Kit
6
Polymerase mixtures for
long PCR (3-10 kb):
Platinum® Taq
13
DNA Polymerase
High Fidelity†
* The use of Platinum® Pfx DNA polymerase requires a PCR purification step prior to cloning.
† These products may be covered by one or more Limited Use Label Licenses (See the Invitrogen catalog or our web site, www.invitrogen.com). By
the use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses.
Table 3 – Sequencing tools from Invitrogen
Kit Name
Application
Template
Page no.
TOPO® Shotgun
Subcloning Kit
Construction of shotgun libraries
for sequencing
BACs, YACs, cosmids, and genomic DNA
14
TOPO® Walker Kit
Determination of unknown gap sequence
Partially sequenced BACs, YACs, and PACs
15
Please visit our web site at www.invitrogen.com/topo
~3~
www.invitrogen.com
TOPO TA Cloning® Kits
Fast cloning of Taq-amplified PCR products
TOPO TA Cloning® Kits
TOPO TA Cloning® Kits combine the TOPO® and TA
Figure 5 - pCR®-TOPO® vectors
TOPO® vectors (Figure 5) are provided linearized with
TOPO
3´-T overhangs and topoisomerase I-activated to readily
SP6
accept PCR products with 3´-A overhangs. This enables
Nsi I
Hind III
Kpn I
Sac I
BamH I
Spe I
BstX I
EcoR I
pCR®II-TOPO®
T
T
recombinants. The
T7
TOPO
fast, 5-minute TOPO® Cloning and yields up to 99%
pCR®-TOPO®
EcoR I
EcoR V
BstX I
Not I
Xho I
Nsi I
Xba I
Apa I
(see box below) technologies to enable fast, effi-
cient cloning of Taq-amplified PCR products. The pCR®-
vectors are ideal for appli-
pCR®2.1-TOPO®
cations such as probe generation, in vitro transcription, or
M13
include:
Hind III
Kpn I
Sac I
BamH I
Spe I
BstX I
EcoR I
TOPO
general subcloning. Some of their convenient features
T
T
lacZα
EcoR I
EcoR V
BstX I
Not I
Xho I
Nsi I
Xba I
Apa I
Cloning®
T7
TOPO
• EcoR I sites flanking the PCR product insertion site for
f1
choice of selection in E. coli
pCR®-TOPO®
3.9 kb
yc i
n
pUC ori
i
or
easy removal of inserts
• Kanamycin and ampicillin resistance genes for your
Am
• T7 (pCR®2.1-TOPO®) or T7 and SP6 (pCR®II-TOPO®)
promoter/priming sites for in vitro transcription
am
• Easy blue/white screening of recombinant colonies
Ka
pic
illin
n
• M13 forward (-20) and reverse priming sites for
TOPO
sequencing or PCR screening
Represents covalently bound topoisomerase I
The consistency and speed of the TOPO TA Cloning® Kits
make them the best way to clone all of your Taq-amplified
PCR products.
Direct ligation with TA Cloning® Technology
The TA Cloning® technology makes it possible to easily clone PCR products produced by Taq polymerase. Taq has a
terminal transferase activity that adds a single 3´-A overhang to each end of the PCR product. TOPO TA Cloning®
vectors contain 3´-T overhangs that enable the direct ligation of Taq-amplified PCR products (Figure 6)(2,3).
Figure 6 - How TOPO TA Cloning® works
1. Add 1 µl of a pCR-TOPO® vector to
1 µl of Taq-amplified PCR product.
2. Incubate for 5 minutes
on your bench top.
3. Transform One Shot®
Competent E. coli.
3´
T
r
Vecto
5´
Topoisomerase I
5´
+
A
5´
3´
Topoisomerase I
T
3´
Topoisomerase I
T
Vecto
r
A
PCR
Product
A
T
Vecto
r
5´
Vector
Activated TOPO TA Cloning® vector
Toll Free: 800 955 6288
3´
A
Taq-amplified
PCR Product
Taq-amplified PCR product
with 3´-A overhangs
Ligation complete. Vector is
ready for transformation
~4~
Topoisomerase I
Zero Blunt® TOPO® PCR Cloning Kits
Simplified blunt-end cloning
Zero Blunt® TOPO® PCR Cloning Kit
TOPO® Cloning to allow easy, high-efficiency cloning of
blunt-end PCR products. The
pCR®-Blunt
II-TOPO®
SP6
vector
(Figure 7) is provided linearized and topoisomerase I-acti-
P lac
TOPO
EcoR I
Pst I
EcoR V
Not I
Xho I
Nsi I
Xba I
Dra II
Apa I
unique Zero Background™ technology (see box below) with
Nsi I
Hind III
Asp718 I
Kpn I
Ecl136 II
Sac I
BamH I
Spe I
EcoR I
Figure 7 - pCR®-Blunt II-TOPO® vector
The Zero Blunt® TOPO® PCR Cloning Kit combines the
T7
TOPO
lacZα
ccd
B
vated so it readily accepts blunt-end PCR products. It
pUC ori
top ligation. The pCR®-Blunt II-TOPO® vector features:
Kanamycin
produces ≥95% recombinants via rapid 5-minute, bench-
pCR®-Blunt IITOPO®
3.5 kb
• The ccdB gene to eliminate background
• EcoR I sites flanking the PCR product insertion site for
Ze ocin
easy removal of inserts
• Kanamycin and Zeocin™ resistance genes for your
TOPO
Represents covalently bound topoisomerase I
choice of selection in E. coli
• T7 and SP6 promoter/priming sites for in vitro RNA transcription and sequencing
• M13 forward (-20) and reverse priming sites for sequencing or PCR screening
The Zero Blunt® TOPO® PCR Cloning Kit offers the easiest,
most effective method available for cloning blunt-end PCR
products.
Eliminate high backgrounds
Due to high background, cloning blunt-end and long PCR products can be difficult and often yields a low percentage of recombinants. Invitrogen’s unique Zero Background™ technology uses the lethal ccdB (control of cell death)
gene to enable high-efficiency cloning. The ccdB gene is incorporated into the cloning site of Zero Background™
vectors. The ccdB protein poisons bacterial DNA gyrase, causing degradation of the
Figure 8 - Positive selection by ccdB
lacZα
ccdB
host chromosome and cell death (4,5).
When an insert is ligated into the vector,
the ccdB gene is disrupted, enabling only
recombinant colonies to grow (Figure 8).
lacZα
ccdB
+
Blunt DNA
If a Zero Background™ vector self-ligates,
the lethal ccdB gene is expressed,
so colonies cannot grow.
By eliminating high vector background,
the Zero Background™ technology yields
Combine a Zero Background™
vector with blunt-end DNA.
lacZα
Blunt DNA
ccdB
nearly 100% recombinants, freeing you
from having to screen hundreds of back-
When an insert is cloned, expression of the
ccdB gene is disrupted, so recombinants can grow.
ground colonies.
~5~
www.invitrogen.com
TOPO® XL PCR Cloning Kit
Efficient cloning of long PCR products
TOPO® XL PCR Cloning Kit
The TOPO® XL PCR Cloning Kit combines TOPO® Cloning,
Figure 9 - pCR®-XL-TOPO® vector
Zero Background™ technology (see box, page 5), and a
unique gel purification step (see box below) to optimize
T
T
earized and topoisomerase I-activated to enable 5-minute,
bench-top ligation and ≥80%
recombinants‡.
Pla
It contains
EcoR I
Pst I
EcoR V
Not I
Xho I
Nsi I
Xba I
Dra II
Apa I
M13
The pCR®-XL-TOPO® vector (Figure 9) is provided lin-
Mlu I
Nsi I
Hind III
Kpn I
Ecl136 II
Sac I
BamH I
Spe I
EcoR I
TOPO
the conditions for cloning long PCR products (3-10 kb).
lacZα
c
cc d
T7
TOPO
B
3´-T overhangs for cloning PCR products produced by
convenient features of pCR®-XL-TOPO® include:
pCR®-XLTOPO®
Kanamycin
pUC ori
most thermostable polymerase mixtures†. Some of the
3.5 kb
• The ccdB gene to eliminate background
Zeocin
• Kanamycin and Zeocin™ resistance genes for your
choice of selection in E. coli
TOPO
Represents covalently bound topoisomerase I
• T7 promoter/priming site for in vitro RNA transcription
and sequencing
• M13 forward (-20) and reverse priming sites for
sequencing or PCR screening
The TOPO® XL PCR Cloning Kit combines gel purification,
TOPO® Cloning and Zero Background™ technology to
enable high-efficiency cloning of long PCR products.
Unique gel purification step improves results
Long PCR often yields multiple products, making
gel purification necessary prior to cloning.
Table 4 - Crystal violet protects long PCR products for safe
gel purification and efficient TOPO® Cloning
Crystal
Violet
Ethidium
Bromide
Total Colonies
(KanR)
275
15
Colonies w/insert
(KanR-AmpR)
258
9
Percent
Recombinants
94%
60%
However, gel purification requires exposure to
ethidium bromide and UV light, which can nick
and damage DNA (6). To protect against nicking,
the TOPO® XL PCR Cloning Kit uses crystal violet to enable visualization of DNA bands in an
agarose gel under ambient light. This eliminates
ethidium bromide and exposure to UV light,
ensuring safe gel purification. Crystal violet
staining results in many more colonies and a
greater percentage of recombinants than using
ethidium bromide and UV light (Table 4).
A 7 kb ampicillin resistance gene sequence was PCR amplified with
Expand™ polymerase. PCR products were loaded onto one gel with
crystal violet and another gel stained with ethidium bromide. The 7 kb
products were gel purified and cloned into the pCR®-XL-TOPO® vector.
The number of recombinants was determined by plating 125 µl of
each transformation on LB plates containing either kanamycin or
kanamycin and ampicillin.
‡ When using chemically competent E. coli, you can expect ≥70% recombinants. For the best results, electrocompetent cells are recommended
because electroporation yields higher transformation efficiencies than chemical methods and does not bias against larger constructs.
† The TOPO® XL PCR Cloning Kit has been tested with Expand™ and eLONGase®.
Toll Free: 800 955 6288
~6~
TOPO® Cloning Kits for Sequencing
TOPO® Cloning vectors for optimized sequencing
Figure 10 - TOPO® Cloning Vectors for Sequencing
The TOPO® Cloning Kits for Sequencing allow fast cloning
and streamlined sequencing of PCR products. The kits
cloning site that positions the T7 and T3 priming sites
M13
T3
TOPO
33 bp
33 bp
Spe I
Sse8387 I
Pme I
EcoR I
73 bp
contain TOPO® Cloning vectors with a minimized multiple
A
Taq-amplified
PCR Product
A
T
only 33 bp away from the PCR product insertion site
M13
pCR®4-TOPO®
M13
pCR®4Blunt-TOPO®
TOPO
M13
T3
33 bp
TOPO
33 bp
EcoR I
Not I
73 bp
Spe I
Sse8387 I
Pme I
EcoR I
insert and less of the vector.
The pCR®4-TOPO vector supplied in the TOPO TA
Cloning®
T7
Blunt-end
PCR Product
(Figure 10). This means you’ll sequence more of your
Choice of vectors
60 bp
T
EcoR I
Not I
TOPO® Cloning Kits for Sequencing
lacZ
cc
dB
c
Pla
Kit for Sequencing has 3´-T overhangs for
pUC o
ri
cloning Taq-amplified PCR products. The pCR®4BluntTOPO® vector supplied in the Zero Blunt® TOPO® PCR
PCR products amplified with proofreading polymerases.
4.0 kb
Each vector is supplied linearized and topoisomerase I-
Am
activated, enabling 5-minute, bench-top ligation and
p ici
T7
TOPO
Kana
my
cin
Cloning Kit for Sequencing has blunt ends for cloning
pCR®4- and
pCR®4BluntTOPO®
60 bp
ll ii n
yielding ≥95% recombinants. Some of their convenient
features include:
TOPO
Represents covalently bound topoisomerase I
• T7 and T3 promoter/priming sites for sequencing and
in vitro transcription/translation
• M13 forward (-20) and reverse priming sites for
sequencing or PCR screening
• The ccdB gene to eliminate background and improve
results (see box, page 5)
• EcoR I sites flanking the PCR product insertion site for
easy removal of inserts
• Unique Sse8387 I site to produce nested deletions for
sequencing internal regions of your insert
• Kanamycin and ampicillin resistance genes for your
choice of selection in E. coli
With their minimized multiple cloning sites, the TOPO®
Cloning Vectors for Sequencing enable effective cloning
and streamlined sequencing for all of your PCR products.
For shotgun sequencing and cloning unknown sequences, refer to TOPO® Cloning Kits for Genomic Analysis,
pages 14 and 15
~7~
www.invitrogen.com
Non-Directional TOPO® Expression Kits
Effective way to express
Non-Directional TOPO® Cloning
Expression Kits
TOPO® Cloning Expression vectors eliminate potential
Using restriction enzymes to clone your gene of inter-
exact DNA sequence you require by performing PCR
est into an expression vector often forces you to com-
with appropriately designed primers. The resulting
promise the final sequence of your insert, especially
recombinant expression vector contains your exact
when there are no useful restriction sites close to your
DNA sequence without any non-coding regions
gene’s coding sequence.
(Figure 11). Non-directional TOPO® Cloning
expression problems by allowing you to insert the
Expression vectors are available in the prokaryotic,
yeast, insect, and mammalian expression systems.
Figure 11 - Comparison of cloning strategies
A. Restriction Digest Method
B. TOPO® Cloning Method
Domain of interest
Domain of interest
RE2
RE1
RE2
RE1
1. Digest with
restriction enzymes
RE1
1. PCR with properly
designed primers
RE2
ATG
Epitope
moter
Pro
A
A
+
moter
Pro
2. Ligate Overnight
3. Transform
RE1
T
T
Epitope
RE2
Epitope
moter
Pro
2. Incubate for 5 minutes
3. Transform
ATG
moter
Pro
Total time: 2 days
Total time: 2 days
RE = Restriction enzyme
= Non-coding sequence
For more information on TOPO® Cloning Expression vectors, see the
Invitrogen catalog or visit our web site at www.invitrogen.com/topo
~8~
Epitope
Total time: <1 day
Directional TOPO® Expression Kits
Time-tested expression results
Directional TOPO® Expression Technology
Directional
TOPO®
With Directional TOPO® Cloning Expression Kits,
Cloning enables you to clone your blunt-
end PCR products in a 5´→3´ orientation into a proven
you will:
• Save time – TOPO® Cloning of your PCR product takes
expression vector using a 5-minute ligation reaction.
just five minutes (Figure 12)
Directional TOPO® Cloning vectors contain a single-strand
• Obtain efficient cloning results - >90% of your recombi-
GTGG overhang on the 5´ end and a blunt end on the
nant clones will be in the correct orientation for expres-
3´ end. The four-nucleotide overhang invades the
sion (Figure 13)
double-strand DNA of the PCR product and anneals to the
• Achieve high-level expression – vectors carry a powerful
CACC sequence that you place in your 5´ primer.
promoter for expression
Topoisomerase I then ligates the PCR product in the
correct orientation.
Figure 12- Time comparison of cloning strategies
Traditional cloning
Directional TOPO® Cloning
PCR
PCR
Restriction enzyme digest on expression
vector and the PCR product
Directional TOPO® Cloning
5 minutes
1 hour
Gel purify DNA fragment.
Dephosphorylate linearized vector
Transformation
13 hours
2 hours
Screen 5 colonies
Overnight ligation
15 hours
12 hours
EXPRESSION
Transformation
13 hours
Total time: 28 hours 5 minutes
Screen 20 colonies
Time Savings:
20 hours
20 hours
EXPRESSION
Total time: 48 hours
Figure 13 – Directional cloning of human open reading frames into pcDNA3.1D/V5-His-TOPO® vector
Clone
No. in correct orientation
No. in reverse orientation
% Correct
18
2
90%
AF016582 (1504 bp)
20
0
100%
AF020833 (1036 bp)
19
1
95%
D32129 (1171 bp)
~9~
Directional TOPO® Expression Kits
High-level protein production in E. coli
Champion™ pET Directional TOPO®
Expression Kits
Champion™ pET vectors are powerful E. coli expression
vectors that use the highly-efficient T7 RNA polymerase to
Figure 14 – Strong expression in pET Directional TOPO® vectors
1
2
3
4
5
6
7
8
I
U
I
U
I
9
10
β-gal
achieve strong transcription levels and high protein yields.
T7 RNA polymerase is expressed by host E. coli under the
control of the IPTG-inducible lacUV5 promoter. This
U
allows you to regulate transcription with IPTG. The additional lacO element found in the T7lac promoter used in
the pET vectors further reduces the basal expression levels while maintaining strong transcriptional activity upon
induction with IPTG. Reported yields of recombinant
proteins from the pET vectors are typically in the range
of tens to hundreds of milligrams per liter of
culture (Figure 14). The pET Directional TOPO®
Expression Kits offer:
• Advanced cloning technology
I
U
U
I
U=Uninduced I=Induced
The lacZ gene was cloned directionally into pET100/D-TOPO®, pET101/D-TOPO®,
and pET102/D-TOPO® vectors and cloned using the restriction digest method into
pET15b and pET32a vectors. Constructs were transformed into BL21 Star™(DE3)
E. coli. A single colony from each transformation was used to inoculate 1 ml LB
medium supplemented with 100 µg/ml ampicillin. Induction with 1 mM IPTG was
performed at OD600=0.5. Two and one-half hours post induction, cultures were
harvested by centrifugation. Pellets were resuspended in 300 µl sample buffer. Ten
microliters of each sample was analyzed on a 4-20% Novex® Tris-Glycine gel.
Note: pET15b contains an N-terminal 6xHis tag while pET32a contains
an N-terminal thioredoxin fusion and a C-terminal 6xHis tag.
Lanes 1 and 2: pET15b/lacZ
Lanes 3 and 4: pET101/D-TOPO®/lacZ
• High-level and regulated expression
Lanes 5 and 6: pET100/D-TOPO®/lacZ
Lanes 7 and 8: pET102/D-TOPO®/lacZ
Lanes 9 and 10: pET32a/lacZ
• Multiple purification, detection, and cleavage options
BL21 Star™: for highest expression
BL21 Star™(DE3) One Shot® Competent E. coli is designed to give you significantly improved expression levels of
recombinant protein. BL21 Star™ is the only strain that contains a mutation in the endonuclease RNase. This mutation
causes less RNA degradation and as a result more RNA is available for translation. With BL21 Star™ you can get up to a
ten-fold increase in protein production. Use BL21 Star™ with any T7 expression system. For added convenience
BL21 Star™ are available in the convenient, cost-effective One Shot® format.
Product
BL21 Star™ One Shot® Chemically Competent E. coli
Efficiency
8
1 x 10
Quantity
Cat. no.
20 x 50 µl
C6010-03
BL21-AI™: for tightly regulated, highly inducible expression
BL21-AI™ One Shot® Competent E. coli is an arabinose-inducible strain designed to give you the maximum expression
with the tightest regulation available from a T7 expression system. It’s the only strain that gives you both tight regulation
and high yields, making it great for high-level expression of toxic proteins. Because it has the tightly regulated arabinoseinducible araBAD promoter upstream of the T7 RNA polymerase gene, you can use it with any T7 promoter-based vector.
All that in the convenient, cost-effective One Shot® format.
Product
BL21-AI™ One Shot® Chemically Competent E. coli
Toll Free: 800 955 6288
Efficiency
8
>1 x 10
~ 10 ~
Quantity
Cat. no.
20 x 50 µl
C6070-03
Directional TOPO® Expression Kits
High-level, constitutive mammalian expression
pcDNA™3.1 Directional TOPO® Expression Kit
Figure 15 – pcDNA3.1D/V5-His-TOPO® Vector
The pcDNA3.1™ Directional TOPO® Expression Kit offers
TOPO
following features (Figure 15):
BGH pA
V
mammalian cell lines with Geneticin® selection agent
p U C ori
pA
40
SV
l li n
5.5 kb
• Neomycin resistance gene for selection of stable
Neomy
cin
A m pi c i
nant protein and rapid purification using ProBond™ resin
pcDNA3.1D/
V5-His-TOPO®
ri
40 o
SV
in a wide range of mammalian cell hosts
• C-terminal V5-His epitope for easy detection of recombi-
pcDNA3.1D/V5-His-TOPO® vectors, expression of lacZ
Stop
P CM
• The CMV promoter for high-level constitutive expression
To demonstrate the high-level expression achieved with
6xHis
Pme I
V5 epitope
Age I
ucts. The pcDNA3.1D/V5-His-TOPO® vector contains the
TOPO
EcoR V
BstX I
Not I
Xho I
Xba I
Apa I
Sac II
T7
Hind III
Asp718 I
Kpn I
BamH I
efficient and directional cloning of blunt-end PCR prod-
Figure 16 – Expression of β-galactosidase from
pcDNA3.1D/V5-His-TOPO®
1
from the vector was compared to pcDNA™3.1/V5-His/lacZ
2
3
The lacZ gene was PCR amplified and
cloned into pcDNA3.1/V5-His-TOPO© and
lacZ pcDNA3.1D/V5-His-TOPO® directional
(Figure 16). The results show that the equivalent expres-
vectors. These constructs were each transfected into 7.5 x 104 COS-7 cells using the
calcium phosphate method. Forty-eight
hours post transfection, cells were harvested. Ten micrograms of total protein
was loaded on each lane of an SDS-PAGE
gel. Expression was analyzed by western
blot using an anti-β-gal antibody. Lane 1:
Mock; Lane 2: pcDNA™3.1/V5-His/lacZ;
Lane 3: pcDNA™3.1D/V5-His/lacZ.
sion levels are achieved.
High-level, stable expression in mammalian cells
ViraPower™ Lentiviral Expression System
Figure 17 – pLenti6/V5-D-TOPO® Vector
stable gene expression and reproducible delivery to both
TOPO
CCC TT
GGG AAG TG G
AAG GGC
TTC CCG
Xho I
Apa I
Sac II
Sfu I
Lentiviral Expression System provides
BamH I
Spe I
BstX I
The
ViraPower™
V5 epitope
Stop
TOPO
dividing and non-dividing cells. You can use the
PSV4
0
V
P CM
RR
ψ
’L
P RSV/5 TR
• CMV promoter for high-level expression
pLenti6/V5D-TOPO®
7.0 kb
LT
RR
pU
• C-terminal V5 tag for convenient detection
C
• Blasticidin resistance gene for fast, efficient stable selection
i
Ampicillin
• Components for efficient packaging of your gene of
pA
40
SV
or
∆U3
/3’
Expression Vector (Figure 17). The vector contains:
idin
stic
Bla
5-minute TOPO® Cloning reaction to prepare your Lentiviral
EM
E
7
pLenti6/V5-D-TOPO® vector to take advantage of a simple,
Figure 18 - Lentivirus readily transduces non-dividing cell types
interest
A
B
The ViraPower™ Lentiviral Directional TOPO® Expression
System provides you with the high levels of stable gene
expression necessary for valid results in virtually any cell
line (Figure 18).
Primary fibroblasts
Adult rat hippocampal
neurons
Contact-inhibited, non-dividing, quiescent primary human foreskin
fibroblasts (A) or adult hippocampal neurons (B) were transduced with
pLenti6/CMV/V5-GFP at an MOI of 5 or 1, respectively. GFP was detected 48 hours post-transduction by fluorescence microscopy.
~ 11 ~
www.invitrogen.com
Directional TOPO® Expression Kits
Quick and easy way to enter the Gateway® System
Gateway® entry clones with Directional
TOPO® Cloning
Figure 19 - Gateway™ Directional TOPO® entry vectors
pENTR/SD/D-TOPO®
Two Directional TOPO® entry vectors are available for
creating a Gateway® entry clone. Once you have cloned
gene 10 RBS
AAG GGT
TTC CCA
GGG AAG TGG
your PCR product into an entry vector, it can be rapidly
Asc I
Not I
TOPO
CCC TT
TOPO
shuttled to a wide variety of Gateway® destination vectors
pENTR/D-TOPO®
for your downstream expression and functional analysis
offer the following features (Figure 19):
Asc I
Not I
needs. pENTR/D-TOPO® and pENTR/SD/D-TOPO® vectors
AAG GGT
T TC C CA
CC C T T
GGG AAG TGG
• attL recombination sites for efficient transfer into any
attL1
Gateway® destination vector
att
L2
• Universal M13 sites to facilitate sequencing
• pUC-based ori for high plasmid yields
• Kanamycin resistance gene for selection in E. coli
C ori
pU
pENTR
pcDNA/GW/D-TOPO Vectors
®
2.6 kb
pcDNA/GW/D-TOPO® vectors give you high-level expres-
Kana m
sion in mammalian cells, and allow you to save signifi-
y ci
n
cant cloning and screening time. Once your gene of interest is cloned into the vector, it will immediately express
from the built-in CMV promoter. This powerful promoter
drives high-level constitutive expression in a wide variety
Figure 20 - pcDNA/GW/D-TOPO® expression vectors
of mammalian cells. pcDNA/GW/D-TOPO® vectors offer
the following (Figure 20):
TOPO
Gateway® entry clone
Asc I
Not I
• attB recombination sites for rapid conversion into a
• Universal M13 sites to facilitate sequencing
attB1
• pUC-based ori for high plasmid yields
CM
selection in mammalian cells
V5
ep
V
pe
ito
• Choice of neomycin or Blasticidin resistance genes for
TOPO
attB2
pU C
ori
PS
0
V4
pA
7
40
® )
PO
om
TO ® )
ycin
/ DO
B la
(p c D N A 3. 2 / G W
P
O
s ti c
D-T
id in
(pc D N A 6. 2 / G W /
ne
Toll Free: 800 955 6288
~ 12 ~
EM
SV
TkpA fl origin
a mp
pcDNA/GW/
D-TOPO®
HTP TOPO® Cloning Kits
Easily clone thousands of PCR products
High-Throughput TOPO® Cloning
plates, incubate for only 5 minutes, then transform the
HTP TOPO® Cloning kits couple TOPO® technology to
supplied chemically competent E. coli with a multi-chan-
high-throughput format, enabling you to easily and simul-
nel pipette (Table 5). With the speed and high efficiency
taneously clone thousands of PCR products. With 500
of TOPO® Cloning, you’ll not only get your clones fast,
reactions of
TOPO®
vector supplied in a single tube, you
can quickly set up your TOPO® reactions in multi-well
you’ll get them the first time, eliminating time wasted
repeating unsuccessful reactions.
Table 5 – HTP TOPO® Cloning Kits
Vector*
TOP10 E. coli Format†
Reactions
Cat. no.
pCR®2.1-TOPO®
Bulk
MultiShot™
MultiShot™ StripWell
500
480
480
K4500-500
K4500-480
K4500-05
HTP TOPO TA Cloning® Kit Dual Promoter
pCR®II-TOPO®
Bulk
MultiShot™
MultiShot™ StripWell
500
480
480
K4600-500
K4600-480
K4600-05
HTP TOPO TA Cloning® Kit for Sequencing
pCR®4-TOPO®
Bulk
MultiShot™
MultiShot™ StripWell
500
480
480
K4575-500
K4575-480
K4575-05
pCR®4Blunt-TOPO®
Bulk
MultiShot™
MultiShot™ StripWell
500
480
480
K2875-500
K2875-480
K2875-05
pENTR/D-TOPO®
Bulk
MultiShot™ StripWell
500
480
K2400-500
K2400-480
pENTR/SD/D-TOPO®
Bulk
MultiShot™ StripWell
500
480
K2420-500
K2420-480
Description
HTP TOPO TA Cloning® Kit
HTP Zero Blunt® TOPO® PCR Cloning Kit
for Sequencing
Directional TOPO® pENTR™ Vectors
* For more information on these vectors, see the catalog or visit our web site at www.invitrogen.com/topo
† Bulk (five 5-ml aliquots)
• MultiShot™ (five 96-well plates)
• MultiShot™ StripWell (five stripwell plates)
~ 13 ~
www.invitrogen.com
TOPO® Cloning Kits for Genomic Analysis
Streamlined shotgun subcloning
TOPO® Shotgun Sequencing Kit
Figure 21 – TOPO® Shotgun Subcloning Kit saves over 13 hours
of time versus traditional shotgun method
The TOPO® Shotgun Subcloning Kit is specifically designed to
expedite traditional shotgun subcloning procedures by saving
Hours
both time and effort in each step.
0
2
4
6
8
10
12
14
16
Traditional
Shotgun
Procedure
TOPO® Shotgun technology was built upon examining each step
of a traditional shotgun subcloning protocol and eliminating
tedious steps and lengthy incubations (Figure 21).
TOPO® Shotgun
Subcloning Kit
This kit includes a specialized vector with numerous features to
make shotgun subcloning easier than ever before. The TOPO®
Shotgun Subcloning Kit includes pCR®4Blunt-TOPO® vector
Shear DNA
(Figure 10, page 7) that allows you to:
Repair DNA and
Dephosphorylate
• Easily construct shotgun libraries—readily accepts blunt-
Gel Purify
Clone
ended DNA fragments. Cloning takes just 25 minutes.
• Eliminate multiple inserts—only vectors containing single
inserts will circularize and propagate.
The TOPO® Shotgun Subcloning Kit utilizes a nebulizer—
• Keep background low—expression of a lethal ccdB gene
a small plastic device used to atomize liquids—and
ensures only recombinant clones will grow (page 5).
compressed air to shear large DNA into 2 kb fragments
• Streamline sequencing—increase efficiency by reading more
suitable for cloning into the pCR®4Blunt-TOPO® vector.
insert and less vector.
Toll Free: 800 955 6288
~ 14 ~
TOPO® Cloning Kits for Genomic Analysis
The fast way to fill in sequence gaps
TOPO® Walker Kit
need to carry out cloning experiments that can add addi-
The TOPO® Walker Kit eliminates the need for
tional days to your experiment. The TOPO® Walker Kit
hybridization-based library screens when isolating
saves you time by:
unknown DNA sequences. Instead, the kit uses
• Ligating a topoisomerase-activated linker to the
unknown end of a DNA fragment in just 5 minutes.
a simple 5-step protocol to save time in your
chromosome walking experiments (Figure 22).
• Using PCR to rapidly amplify the unknown sequence
Once the unknown DNA fragment is amplified, the PCR
• Sequencing the PCR product directly—no subcloning
steps are required
product can be sequenced directly. There’s no
Figure 22 – How TOPO® Walker works
Step 1
P
3
5
ACGT
1. Digest complex DNA
(such as BAC, YAC,
or PAC) to completion
with an enzyme that
leaves a 3´ overhang.
TGCA
5
3
P
Step 2
5
3
3
5
2. Dephosphorylate the
DNA to allow ligation
of the TOPO® Linker.
TGCA
ACGT
Step 3
3
5
GSP1
ACGT
3. Primer extend with
TGCA
Taq DNA polymerase
and a gene-specific
primer (GSP1).
A
3
5
Step 4
5
A
5 TTCCC
GSP1
ACGT
3
TOPO® Linker
4. Ligate the TOPO®
3
5
Linker to the DNA.
TOPO
Step 5
GSP1
ACGT
GSP2
A
T
3
TOPO® Linker
5
3
5. Perform PCR using a second
gene-specific primer (GSP2) and a
primer complementary to the TOPO®
Linker (LinkAmp Primer 1 or 2).
5
LinkAmp Primer 1 or 2
Known DNA
Unknown DNA
PCR makes it easy — TOPO® Technology
makes it fast
which topoisomerase I is covalently bound. In just
The key to the speed of the TOPO® Walker Kit is
3´-A overhang of your DNA sequence. Subsequent
the
TOPO®
Linker (Figure 23). The
TOPO®
Linker is
5 minutes you can ligate the TOPO® Linker to the
PCR using a primer specific to the linker and a
a 58 bp, double-stranded DNA sequence containing
gene-specific primer from your known sequence
two PCR primer sites, and a 3´- T overhang in
amplifies the sequence gap.
Figure 23 – The topoisomerase-activated TOPO® Linker
LinkAmp Primer 1
LinkAmp Primer 2
TAGAAGGCACAGTCGAGGACTTATCCTAGCCTCTGAATACTTTCAACAAGTTACACCCTT
AAAAAAAATCTTCCGTGTCAGCTCCTGAATAGGATCGGAGACTTATGAAAGTTGTTCAATGTGGGA
P
~ 15 ~
TOPO
TOPO
Represents covalently bound topoisomerase I
www.invitrogen.com
TOPO® Cloning Technology
Custom TOPO® Cloning Adaptation Service
The development of gene-based therapeutics and diagnostics products requires the rapid analysis of a vast number
of gene sequences. When screening gene targets that are
of commercial importance, being the first to identify,
clone, express, and validate these genes is crucial.
Invitrogen’s Custom TOPO® Cloning Adaptation Service
puts the power of TOPO® Cloning into your vector.
With your own vector adapted to TOPO® Technology, you can:
• TOPO® Clone your favorite vector – you won’t have to
compromise on vector features to meet your needs
• Save time – TOPO® Cloning takes only 5 minutes and is
so effective, you won’t have to repeat experiments
• Maintain your current experimental strategy – adapting
your own vector for TOPO® Cloning doesn’t change your
downstream studies, but it will get you there faster
Your results are guaranteed
Every TOPO® Cloning Kit is functionally tested to ensure
polymerase. The size of the PCR product is verified and
that you get successful results. To test each kit, a control
TOPO® Cloned. The resulting percent recombinants must
DNA fragment is PCR amplified with the appropriate DNA
meet our stringent standards in order to pass quality control.
Clone it today
With unrivaled consistency and speed, TOPO® Cloning Kits
reactions all in the same day. And up to 99% recombinants
are the most effective way to clone PCR products.
ensures that you’ll get your clones the first time, every time.
The rapid 5-minute, bench-top ligation enables you to
For PCR cloning results you can count on, order a
perform your PCR, TOPO® Cloning, and transformation
TOPO® Cloning Kit today.
References:
1. Shuman, S. (1994) J. Biol. Chem. 269: 32678-32684.
2. Clark, J.M. (1988) Nuc. Acids Res. 16: 9677-9678.
3. Mead, D. et al. (1991) Bio/Techniques 9: 657-663.
4. Bernard, P. and Couturier, M. (1992) J. Mol. Biol. 226: 735-745.
5. Bernard, P. et al. (1993) J. Mol. Biol. 234: 534-541.
6. Rand, K.N. (1996) Elsevier Trends Technical Tips Online.
Important Licensing Information
Performance of the polymerase chain reaction (PCR) is covered by one or more of U.S. Patent Nos. 4,683,202; 4,683,195; and 4,899,818 issued to Cetus Corporation and owned and
licensed by Hoffmann-LaRoche Molecular Systems, Inc. Purchase of any of Invitrogen’s PCR-related products does not convey a license to use the PCR process covered by these patents.
Purchasers of these products must obtain a license to use the PCR process before performing PCR. TOPO TA Cloning® is covered under one or more of U.S. Patent Nos. 5,487,993;
5,766,891; 5,827,657 and corresponding foreign patents. For research purposes only. Other patents pending. Products referred to herein which are the subject of one or more of U.S.
Patent No. 5,910,438 and 6,180,407, and/or corresponding foreign patents, are sold under license from the Université Libre de Bruxelles for research purposes only (Limited Use Label
License No. 54: ccdB-Fusion Vectors). Zeocin™ is a trademark of CAYLA. For research use only. Expand™ is a trademark of Boehringer Mannheim Corporation.
710-021849 070803 10M
~ 16 ~
CYAN MAGENTA YELLOW BLACK
DIE MARK
TOPO® Cloning Technology
Fast, effective cloning of PCR products
Direct your cloning future.
TOPO® Cloning Technology
With TOPO® Cloning
Technology You Can:
• Clone Taq-amplified,
blunt-end, and long
PCR products
• Sequence or clone
directly into an
expression vector
• Ligate in 5 minutes at
room temperature and
obtain up to 99%
recombinants
These products may be covered by one or more Limited Use Label Licenses (See the Invitrogen catalog or our web site,
www.invitrogen.com). By the use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses.
For research use only. Not intended for any animal or human therapeutic or diagnostic use. Printed in the U.S.A. ©2003 Invitrogen
Corporation. All rights reserved. Reproduction forbidden without permission.
Corporate headquarters:
1600 Faraday Avenue • Carlsbad, CA 92008 USA • Tel: 760 603 7200 • Fax: 760 602 6500 • Toll Free Tel: 800 955 6288 • E-mail: tech_service@invitrogen.com • www.invitrogen.com
European headquarters:
Invitrogen Ltd 3 • Inchinnan Business Park • Fountain Drive • Paisley PA4 9RF, UK • Tel: +44 (0) 141 814 6100 • Fax: +44 (0) 141 814 6260 • E-mail: eurotech@invitrogen.com
710-021849 070803
10M
• Obtain up to 99% recombinants
• Ligate in 5 minutes at room temperature
• Clone Taq-amplified, blunt-end, and long PCR products
With TOPO® Cloning Kits You Can: