Biomedical Engineering Reference
In-Depth Information
5. Visualize the resolved product with a UV transilluminator or a
gel imaging system. A prominent and unique band at the
desired position of the length of the insert should be seen.
6. Cut the PCR product band from the gel with a clean sharp
razor and purify the insert DNA using a commercial kit (e.g.,
E.Z.N.A. gel extraction kit). Measure the concentration of the
purifi ed insert DNA with the NanoDrop spectrophotometer.
1. Design PCR primers for the vector so that they hybridize with
the ends of the insert DNA. The hybridizing parts can either
be already incorporated in the vector or be added in this PCR
step as overhangs in the primers. The homolog overhangs can
range from 0 to 40 bp in order to make the annealing tempera-
ture of the hybridizing sequence between vector and insert to
be over 55 °C.
2. Set up the PCR on ice as tabulated below:
3.2 Preparation
of Linear Vector
Volume per
50
Final amount in
50
Initial concentration
μ
L reaction
μ
L reaction
Phusion PCR buffer (5×)
10
μ
L
1×
dNTP mix (40 mM)
1
μ
L
0.8 mM
Phusion High-Fidelity DNA
polymerase (2 U/
0.5
μ
L
1 U
μ
L)
Forward primer (10
μ
M)
2.5
μ
L
0.5
μ
M
Reverse primer (10
μ
M)
2.5
μ
L
0.5
μ
M
Vector DNA template (variable)
Variable
1 pg-10 ng
Nuclease-free water
Up to 50
μ
L
3. Run the PCR with the following conditions. Again, for the
Phusion enzyme, the annealing temperature should be 3 °C
higher than the lower
Tm
of the two primers;
Tm
should be
calculated using the part of primer that hybridizes with the
insert and using the nearest-neighbor method [
7
].
Cycle number
Denature
Anneal
Extend
1
98 °C, 30 s
2-31
98 °C, 10 s
(
Tm
+ 3) °C, 30 s
72 °C, 15 s/kb
32
72 °C, 5 min
4. Combine 50
L of 6× DNA
loading dye and run the product on a 1-1.5 % percent agarose
gel. The time for electrophoresis is dependent on the length of
the product.
μ
L of the fi nished reaction with 10
μ
