Hi all, I am planning to PCR a 7430 bp fragment from a big plasmid(11 kb) with Thermo Phusion hot start II DNA pol. Thermo recommend to use 15s-30s as extension time per 1 kb length. Overall GC% of my amplicon is 51% and primers GC% and Tm are 46-47% and 58-59C. Is 3 min extension time too long for 7430 bp specially with phusion hot start II ?I also checked PCR fidelity and it is 8% as error percentage which in my knowledge is not bad(I might be wrong).What is the ideal extension time I should use to PCR 7430 fragment from a pure plasmid?
A 3:00 extension time is not too long. At 30 seconds per kb, you could use a 3:30 extension time. Your product isn't that big. You might need to linearize your plasmid to eliminate supercoiling. Make sure the enzyme doesn't cut in your desired PCR product. But you can try adding undigested plasmid first and see if you get amplification.
Try out 3:30 and hopefully it will give you good amplification of your target gene.
Good luck!
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