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Primer mismatch bother me! - when enzyme sites added at 5' ends, mismatch increases. (Jan/06/2008 )

Dear all,

Before enzyme sites added, I amplified the fragments from the plasmids, I could get the bright clear target fragment, but the second step PCR adding enzyme sites at 5' ends of the primers, the results:

I could got the target fragment band, which was brighter than the backgroud, but also with smeared product, increased the annealing T, the result was the same.

-genejackie-

Can we have an image, please?

-swanny-