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PCR - 2nd reamplification does not work (May/07/2004 )

blink.gif hi everyone,
I have this really weird problem, and hope someone could help me out.

I am trying to reamplify my PCR product (4.5kb) after adding restriction sites to the ends (NotI/FseI) -- however, it fails every time I try to reamplify.

The long story:
I have this DNA template, made from 3 fragments of DNA by overlap/extension method. My end product is 4.5kb. I sequenced it and checked for appropriate overlapping. That's went well. This 4.5kb product can be reamplified very well. Next, I wanted to add 2 restriction sites to both ends by using primers with overhang restriction sites. This step worked fine too. So now I have a product with restriction sites added. However, when I take this product with restriction sites added (NotI-Template-FseI) and try to reampify it using the same pairs of primers I initially used to get this product -- PCR failed, and I got a smear! I am not sure what is going on. I checked and played around the Tm, PCR cycles, and even purification of templates. I noticed there are potential hairpin formation in both the product and the primer pair (4bp), but I don't think that's an issue. If hairpin formed in the primer pair then I would not have been able to add restriction sites from the beginning. Potential hairpin formation in templates at the termini is very unlikely, and does not seem to be a problem (i don't think) -- Still, PCR does not work!

Please provide some ideas if you could --- Thanks!



Smears frequently result from the use of too much input DNA template and/or too many cycles. When I reamplify, I dilute 1:100 and add 10 uL to a 125 uL reaction. Amplification is for only 9 cycles.


Can you fill up the NotI overhang with T4 DNA pol and see if that changes product output quality? I know it defeats the purpose of the vector but it is crucial to see if the NotI site you added is the source of the problem.