Primer mismatch bother me! - when enzyme sites added at 5' ends, mismatch increases. (Jan/06/2008 )
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.
Can we have an image, please?