I want to do random mutagenesis in 5 kb fragments. I want to use the product to do directed evolution. I used two error-prone PCR kits (Aligent and Takara) and can't amplify fragments which size is 5kb. I tried nine primers and my primers are correct by verified with Taq polymerase. So, I'm confused. Can I use error-prone PCR to do random mutagenesis for 5kb fragment?
Does the kit say this polymerase is fit for such long PCR> Longer than 1KB PCRs are always finicky to tweak. I would recommend doing it sequentially may be. We tried multiple kits for amplifying a 3kb fragment but it was giving us troubles. Although all the kits claimed that long PCRs can be done.
Mahmudul Hasan The kit mentioned that it can amplify and do mutagenesis fragments below 6kb. I agreed with you that maybe length of fragments is the key problem.
I used multiple polymerases from reputed companies that claimed to be able to amplify 10kb but couldn't do 3 kb.
Those claims are usually for ideal scenarios, low GC rich, fewer repeats, etc. Let me know how you solved your issues.
Mahmudul Hasan I didn't solve my problem actually. I have changed method. I gave up use error-prone PCR and used EMS to do mutagenesis and succeeded.
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