Biomedical Engineering Reference
In-Depth Information
Table 1
PCR mix
Template DNA
0.5
μ
L (1-5 ng)
10× CxL polymerase buffer
5
μ
L
dNTP mix (2.5 mM each)
4
μ
L
Primer-1 (10
μ
M)
4
μ
L
μ
μ
Primer-2 (10
M)
4
L
Proof-reading X-7 polymerase [
30
]
1
μ
L
ddH
2
O
31.5
μ
L
Total
50
μ
L
Table 2
PCR program
98 °C
2 min
98 °C
15 s
57 °C
15 s
30 cycles
72 °C
2 min (1 kb/30 s)
72 °C
3 min
4 °C
∞
3.2 Creating DNA
Fragments Through
PCR
1. PCR amplify (
see
Tables
1
and
2
), with primers containing a
uracil base, the desired feature from plasmids or other DNA (
see
Note 11
). This enables creation of long single-stranded 3
ends
for ligation-independent cloning. Use a proof-reading poly-
merase compatible with uracil in the primers, e.g., the commer-
cial
PfuTurbo
®
Cx Hotstart DNA Polymerase (Agilent) or
preferably the proof-reading X-7 polymerase [
30
] (
see
Note 12
).
2. Add 1
′
L of DpnI to each of the PCRs and incubate for 1 h at
37 °C, followed by 20 min of heat inactivation at 80 °C
(
see
Note 13
).
3. Prepare a 1 % agarose gel in 1× TAE buffer for gel electropho-
resis (
see
Note 14
). Pour the gel in a gel tray prepared with
comb. The gel should be hardened within 30 min and the
comb can be removed. Place the gel tray with gel in a gel
chamber and over with 1× TAE buffer.
4. Mix 2
μ
μ
L of each DpnI-treated PCR product with 2
μ
L ddH
2
O
and 1
L 5× GelRed™ and load into the wells of the prepared
gel. Load 5
μ
L of 1 kb Plus DNA Ladder into a neighboring
well and perform the gel electrophoresis at 100 V for 25 min.
μ
