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 linear vector should be seen.
6. Cut the PCR product band from the gel with a clean sharp
razor and purify DNA using a commercial kit (e.g., E.Z.N.A.
gel extraction kit). Measure the concentration of the purifi ed
vector DNA with the NanoDrop spectrophotometer.
1. Set up the cloning reaction on ice as below. The amount of insert
required will be dependent on its size and should be calculated to
maintain an insert:vector molar ratio between 1:1 and 2:1.
3.3 CPEC
3.3.1 CPEC
of a Single Insert
Volume per
20
Final amount per 20
μ
L
Initial concentration
μ
L reaction
reaction (
see
Note 3 )
Phusion HF buffer (5×)
4
μ
L
1×
dNTP mix (40 mM)
0.4
μ
L
0.8 mM
Phusion High-Fidelity DNA
polymerase (2 U/
0.2
μ
L
0.4 U
μ
L)
Vector DNA (variable)
Variable
50-100 ng
Insert DNA (variable)
Variable
-
Nuclease-free water
Up to 20
μ
L
2. Run the CPEC reaction using the following conditions
(
see
Note 4
) (Fig.
3
):
Cycle number
Denature
Anneal
Extend
1
98 °C, 30 s
2
98 °C, 10 s
(
Tm
+ 3) °C, 30 s (
see
Note 5
)
72 °C, 15 s/kb (
see
Note 6
)
3
72 °C, 5 min
1. Set up the cloning reaction on ice as below. The amount of insert
required will be dependent on its size and should be calculated to
maintain an insert:vector molar ratio between 1:1 and 2:1.
3.3.2 CPEC
of a Single Library
Volume per
20
Final amount per 20
μ
L
Initial concentration
μ
L reaction
reaction (
see
Note 3 )
μ
Phusion HF buffer (5×)
4
L
1×
dNTP mix (40 mM)
0.4
μ
L
0.8 mM
Phusion High-Fidelity DNA
polymerase (2 U/
0.2
μ
L
0.4 U
μ
L)
Vector DNA (variable)
Variable
50-100 ng
Insert library DNA (variable)
Variable
-
Nuclease-free water
Up to 20
μ
L
