never get bands by pfu related enzymes - (Jul/10/2010 )
i'm going to do a fusion pcr.there is a 1.6 kb to be fused with another 1.6 kb .i never get this 1.6 band with pfu while taq amplify it on my template very well.i have changed my primers three times and also my enzyme.pfu easily amplifies b2m gene on my template(3kb
)while this 1.6 kb high gc content region will fail in pcr.what is ur suggestion to overcome this nerve-racking problem?if taq adds a at the end what should be done to remove them?
thanks a lot
I also had the problem that some seq just couldn't be amplified with pfu. Don't know why, never found out. If you want you can try to mix taq & pfu, my bench neighbor had good results with that method.
What are your downstream applications for the pcr-product? You can insert restriction sites if your worried about additional a's.
snoopyx on Jul 12 2010, 02:36 AM said:
What are your downstream applications for the pcr-product? You can insert restriction sites if your worried about additional a's.
hi
thanx for ur reply. i thought its only me that has this strange problem!


thanx everybody for participating in this forum i 'm glad to hear your experiences about this kind of pcr and pfu enzyme