Hi,
I'm working on introducing single point mutations with mostly overlapping primers I designed using the QuickChange XL manual, and doing the PCR with phusion polymerase. My plasmid is quite big at 10 kb, and as the Tm of the primers is all above 78, I have been using a two-step PCR (no separate annealing step). I'm using 40sec/kb for the elongation at 72C.
However, when I run my PCR products on a 1% gel, I am seeing streaking on the top half or so of the gel, as well as many primer dimers at the bottom. I've tried adjusting MgCl2 concentrations, as well as DMSO, number of cycles, elongation time, and different annealing conditions. Each time I either have the streak or no amplification. I've attached an image of a gel with different mutations under the same conditions to show the streaking.
Has anyone had a similar problem? What did you do?
Thanks!
Sometimes when a pcr will not work well then first amplification of a slightly larger product then nested amplification of the first round product can often produce a clean product where simple amplification off the much larger target of the whole genome works poorly
What is the annealing temperature you are using? I suggest , you to do gradient PCR between 50-70 degrees. Also, filter your dd water before use.
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