I would like to perform an overlap PCR. I was able to synthetize one of fragments but not the other one. I tried it with Pfu and Phusion polymerase as waell. Although worked with Taq (annelation: 58 C, but didn't with HF Taq), I have to use an high-fidelity polymerase.
Sebastián Dupraz
Hi Zsolt,
Would you share a bit more regarding the conditions you tried do far, the source of your templates as well as the length, melting temperature and general construction of the primers?
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Laurence Stuart Dawkins-Hall
Commence your PCR with 1 x denaturation cycle of 96C for 5 min Also you could try adding 5 more cycles to your PCR; particularly if your PCR currently has 25 or 30 cycles
Dear Zsolt
- Regarding the PCR fragment that did work with Taq but didn't work with pfu try a range of annealing temp:
- In particular verify the Tm of your primers using a program like IDT Oligo analyser:
- https://eu.idtdna.com/calc/analyzer
- And go from Tm-3C to Tm +1/2C in 1c increments for your annealing temp
- In other words set up 5-6 separate PCR reactions where you vary the annealing temp by Tm-3; Tm-2 etc....Tm+1/2C
- Sometimes the optimal annealing temp of classical Taq will vary from that of newer PCR polymerases like Phusion or modern PFU
- Regarding the actual OCR set up in my experience with both PFU and phusion standard protocols recommended by the manufacturer tend to work well
- Note: these programs tend to have much shorter duration times for denaturation annealing and extension/strand replication than Taq as they are much more processive enzymes
- Also, if you are not already doing so:
- Regarding your fragment you have yet to amplify apart from the above considerations you should always bear in mind that not all primers work and/or your target sequence might be difficult in terms of high GC or AT content (> 60%) or runs of 4/5 or more GC AT residues which will reduce PCR efficiency; or primer dimers
- Thus, check the composition of your target sequence and also try alternative primers preferably made against your target with a program like primer 3 which will check for dimerisation hair pin loop formation etc
- If you find your target sequence is GC rich add 5% DMSO or 1M betaine to your PCR reaction
- If AT rich try a combined annealing extension temp of 60-65C
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