Circular Polymerase Extension Cloning - DNA Cloning and Assembly Methods - page 110

Fig. 5 CPEC of combinatorial gene libraries. Lanes 2, 3, and 4 show the CPEC
reaction products after 5, 10, and 20 thermal cycles. The upper arrow indicates
the full-length cloning product of a 306 bp gene library and a 741 bp gene library
as well as the vector (2.5 kb); the lower arrow indicates the remaining empty
vector. Lane 1 is the 1-kb ladder from Bio-Rad

3.3.3 CPEC
of Combinatorial
Gene Libraries

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 for each insert library.

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/ μ L)

0.2 μ L 0.4 U

Vector DNA (variable) Variable 50-100 ng

Insert library DNA (variable) Variable -

Nuclease-free water Up to 20 μ L

2. Run the CPEC reaction using the following conditions
( see Note 8 ) (Fig. 5 ):

Cycle number Denature Slow ramp anneal Anneal Extend

1 98 °C, 30 s

2-31 98 °C, 10 s 70 to ( Tm +3) °C

(0.1 °C/s)

( Tm + 3) °C, 2 min
( see Note 5 )

72 °C, 15 s/kb
( see Note 6 )

32 72 °C, 5 min