Overlap extension PCR

Primer Design

15 to 21 bp overlap

no self-pairing and low G/C content.
The length of the overlapping primers: 27 bp to 40 bp, with a minimal overlapping region of 15 bases.
The outer primers for the full-length ORF amplification do not contain any overlapping sequence

Asymmetric  Overlap extension PCR

1. PCR

PCR reaction 1 & 2

1 ng plasmid DNA
0.5 ÎŒL 10 mM dNTP
200 nM Outer primer
20 nM mutant primer (20% or 10% of outer primer)
1 U Phusion
5 ÎŒL 5X Phusion buffer
water to 25 ÎŒL.

2. Mix two PCR reactions, 94 ^oC 1 min, incubate at 60°C for 1 min and 72°C for 5 to 15 min.

3. Gel purify, digestion then transformation.

Fusion of 3 PCR fragments

1. PCR three fragments using Phusion or Deep Vent. Primers should have overlap regions.

2. Fusion

10 ÎŒl ThermoPol reaction buffer
5 ÎŒl 10 mM dNTP mix
20 ÎŒl (100 ng) fragment 1
20 ÎŒl (100 ng) fragment 3
4 ÎŒl (20 ng) fragment 2 (middle one)
2 ÎŒl 100 mM MgSO4
0.8 ÎŒl Deep Vent DNA Polymerase
38.2 ÎŒl H2O

94°C 2 min
94°C 15 s
56°C 20 s
72°C 1 min
10 cycles

3. Amplification

72 ÎŒl H2O,
10 ÎŒl ThermoPol reaction buffer
5 ÎŒl 10 mM dNTP mix
3 ÎŒl of unpurified PCR product from fusion step
2 ÎŒl 100 mM MgSO4 solution
2 ÎŒl F primer
2 ÎŒl R primer (both 2 ÎŒM)
0.8 ÎŒl Deep Vent DNA Polymerase.

94°C 2 min
94°C 15 s
62°C 20 s
72°C 2 min 30 s
35 cycles
72°C 3 min

Fusion of PCR product with Vector without restriction digestion

1. Amplify insert using chimeric primers.

F
NNNNNNNNNNNNNNNNNNNNNNNNXXXXXXXXXXXXXXXXXXXX
NNNNNNNNNNNNNNNNNNNNNNNNXXXXXXXXXXXXXXXXXXXX-xxxxxxxxxxxxxxxxxxxxnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnnnnnnnnnnnnnxxxxxxxxxxxxxxxxxxxx-XXXXXXXXXXXXXXXXXXXXNNNNNNNNNNNNNNNNNNNNNNNN
                                         R 3'XXXXXXXXXXXXXXXXXXXXNNNNNNNNNNNNNNNNNNNNNNNN5'

initial denaturation at 100°C for 2 min
denaturation at 94°C for 30 s
annealing at 60°C for 30 s
extension at 68°C for 90 s/kb for 30–32 cycles, with a final extension of 68°C for 10 min.
Gel purify PCR-amplified insert
The final PCR product has overlapping regions with the vector.

2.  Megaprime PCR
Mix acceptor plasmid vector (3–30 ng) with 250 × molar excess of insert DNA, in  10-ÎŒL total volume containing Phusion DNA polymerase, dNTP and buffer.

PCR
Denature: 98°C for 30 s
annealing: 60°C for 30 s
extension: 98°C for 1.5 min per kb
15 cycles
one extra 10-min extension.

Efficiency: Phusion > KOD  > Pfu

3. Add 10 units Dpn1 directly to PCR reaction, digest at 37 oC for 1 hr.

4. Transform E. coli

Ist round PCR

Run 25 to 35 cycles to check if PCR is going to work

Run 10 cycles, use this reaction as 2nd PCR template

2nd Round PCR

Mix 10 ul first round PCR reaction, add 1 ul two outer PCR primers, add 10XPCR buffer, dNTP, water (to 25 or 50 ul), Pfu or Phusion

Run 20 to 25 cycles

Take 5 ul for gel electrophesis

See if the size is good

Gel purify (if parent vector has the same antibiotics), or purify directly with spin column, elute with 20 ul H2O

Digestion

Digest with restriction enzymes, heat inactivate at 75 oC for 10 min, or purify by spin column

Optionally, the PCR products can be digested with restriction enzymes without purification (using FastDigest buffer) if:

• parent vector has different antibiotics
• a single PCR band

Add gel purified (digested) vector, DNA ratio (1:3 to 1:1, vector:insert, no need to use spectrophotometer for quantitation).

Ligation

Add 2.5 ul ligation buffer and 0.5 ul ligase, ligate 2 hours at RT.

Transformation

Transform 10 ul ligation mix with 50 or 100 ul Top10 or STBL3.

Grow cells O/N at 37 oC

Spin down cells, plate on LB/Amp or LB/Kan