Restriction digestion
DNA concentration
Gel electrophoresis
DNA gel electrophoresis
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When running DNA gels with ethidium bromide the
intesity of the band will reflect the amount of DNA
Nicked
Thus when digesting a DNA sample short
Superfragments will be less intense than longer
coiled
fragments
Supercoiled and open circular DNA molecules
(relaxed plasmids with a nick in one of the strands
in the DNA double helix) move differently in the
gel compared to linear fragments
The percentage of agarose can be optimized for
identification of shorter or longer fragments
Normal yield for a plasmid miniprep 300-400 ng/µl
Measuring DNA
We will be using a Nanodrop instruent to
measure DNA concentrations. Use 1 ml of
your plasmid prep.
 A solution containing 50 µg/ml of double
stranded DNA has an absorbency (optical
density) of 1.0 at a wavelength of 260 nm.
50 µg/ml /1.0 = [DNA]/OD read
[DNA] = 50 µg/ml x OD read
 DNA mg/ml = ODA260read x 50 µg/ml. If
you have diliuted the DNA you have to
multiply with the dilusion factor
 A260/230 should be around 1.8 for pure DNA
and around 2 for pure RNA
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Measuring DNA
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DNA quality measurement is based on the fact
that OD at 260 nm is twice that at 280 nm and
230 if the solution contains pure DNA. If there is
a contaminant, OD ratio decreases.
Clean DNA has a ODA260 /ODA280 and a ODA260
/ODA230 between 1.8 and 2.0, if value is smaller
it indicates a contamination.
pCR 2.1-TOPO
909
Primers for sequencing:
M13F and M13R
763
Restriction of clones SUVR1
NcoI (4601)
EcoRI (4414)
DdeI (4240)
NcoI (4134)
Restriction of clones SUVR1 RC
Restriction of clones SUVR2
Restriction of clones SUVR2 RC
Restriction of clones SUVR4
PstI (17)
a tsul2 -9 75 R(4 66 3 )
PstI (4555)
T7 prom ote r
XbaI (53)
S 4 SE x on
T7 prim e r
a tsul2 -1 63 L(3 93 2 )
M 13 (-2 0 ) forw ard prim e r
M 13 re v ers e prim e r
M 13 (-4 0 ) forw ard prim e r
la c re pres s or binding s ite
f1 origin
la c promote r
Ka n promote r
Clone pCR2.1-TOPO,PCR Product of SUVR4full-deltaEx
PstI (1207)
4663 bp
Ka n(R)
ApaLI (3199)
NcoI (1586)
pUC origin
ApaLI (1953)
Amp(R)
Restriction of clones SUVR4 RC
atsul2-163L
EcoRI (8)
S4SExon
PstI (17)
PstI (4045)
atsul2-975R
T7 promoter
AvaI (41)
EcoRI (3921)
T7 primer
BamHI (3890)
M13 (-20) forward primer
HindIII (3872)
M13 (-40) forward primer
M13 reverse primer
f1 origin
lac repressor binding site
Kan promoter
lac promoter
Clone pCR2.1-TOPO,PCR Product of SUVR4full-deltaEx-RC
PstI (1207)
4663 bp
Kan(R)
ApaLI (3199)
NcoI (1586)
pUC origin
ApaLI (1953)
Amp(R)
Restriction of clones ASHH2
Restriction of clones ASHH2 RC
Restriction of clones - ASHR3
EcoRI (8)
PstI (17)
(4825)
NcoI (4241)
(4660)
ApaLI (4025)
AvaI (41)
T7 promoter
(4595)
ApaLI (3960)
T7 primer
EcoRI (3509)
M13 (-20) forward primer
BamHI (3478)
M13 (-40) forward primer
HindIII (3460)
ApaLI (360)
SP6 promoter
ccdB (no ATG)
SP6 primer
M13 rev erse primer
lac repressor binding site
Kan promoter
ASHR3 RC
Clone pCR-Blunt II-TOPO,ASHR3-4010-RC
4866 bp
4265 bp
lac promoter
Kan(R)
NcoI (1462)
ApaLI (2745)
bla promoter
NcoI (1901)
pUC origin
AvaI (1981)
ApaLI (2248)
XmaI (1991)
AvaI (1991)
Restriction of clones - ASHR3 RC
EcoRI (8)
PstI (17)
ApaLI (3822)
(4234)
AvaI (41)
(4169)
ApaLI (3757)
T7 promoter
(3963)
NcoI (3541)
T7 primer
EcoRI (3509)
M13 (-20) forward primer
BamHI (3478)
M13 (-40) forward primer
HindIII (3460)
ApaLI (360)
SP6 promoter
ccdB (no ATG)
SP6 primer
M13 rev erse primer
lac repressor binding site
Kan promoter
ASHR3 RC
Clone pCR-Blunt II-TOPO,ASHR3-4010
rc-RC
4866
4265
bpbp
lac promoter
Kan(R)
NcoI (1462)
ApaLI (2745)
bla promoter
NcoI (1901)
pUC origin
AvaI (1981)
ApaLI (2248)
XmaI (1991)
AvaI (1991)
SmaI (1993)
Which restriction enzyme to use?
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Different enzymes require different buffers
When making double digests you might have to
compromise on the buffer you use so it is
optimized for both enzymes, this may require
using a buffer that does not provide 100%
activity for both enzymes.
In your lab journal you should write the expected
fragment sizes and in which orientation your
gene is cloned based on the results you get.
Restriction of clones SUVR2 G
Restriction of clones SUVR2 G RC