I am getting 3PCR products of different sizes when amplifying off a plasmid, and I think it is due to unintentional primer binding. I am amplifying a trimer off the plasmid, and the 3 sizes correspond to the monomer, dimer, and trimer. My primers bind to the parts of the plasmid flanking the trimer, so they each only bind to the plasmid once. The primers have overhangs so the PCR product can be used in Gibson Assembly, and I am thinking that the overhangs may be close enough in sequence to bind somewhere on the trimer. Are there any tools to check primer specificity, or do you have any suggestions about what else could be going wrong?
depending upon the host strain the plasmid was isolated from, you might also have some low level recombination occurring if you had exact trimers in the plasmid. So the population of plasmids in your template prep might include monomers, dimers and trimers and hence giving you those products.
However if you really think it is primer binding to other sites, you might try raising the stringency of the annealing by increasing the annealing temperature during the PCR by a few degrees to see if that helps.
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