I am trying to do PCR of mouse pluripotent genes, but after running the gel primer dimers are prominent. anybody please let me know the solution.
Increase the primers length if possible and redesign the primers again with optimum requirements.
Redesign your primers and run a PCR in silico to check they do not dimerise. I use a freeware program called serial cloner.
http://serialbasics.free.fr/Serial_Cloner.htm
Other than that optomise your annealing temperatures by performing a gradient PCR.
Hi Digvijay
I usually do three things to reduce sec. structures:
-Temperature gradient for T optimization;
-Reducing the primer concentration;
-Adding a time increment can also help. In addition, you can also try to adjust your MgCL2 concentration to increase the specificity of your reaction.
Good luck, I hope some of this helps,
D
1: Reduce primer concentration, and optimize by Temp gradient reaction (see Dusan)
2: Design a new set of primers with better algorithms that address dimer issues.
3: Search if someone else has published a primer set that has worked fine earlier for your target template.
I concur with previous answers. If problems persist following optimizations you could additionally try touchdown PCR. I assume you have already tried HotStart PCR.
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