I was doing amplification using mutagenic primer, and the result of the PCR in polyacrylamide gel showed two bands. Both were significant bands. I have increasing the annealing temperature and increasing the MgSO4 volume but it did not work. But when I used normal primer, it showed only one band. But the restriction enzyme I have is the one which only can digest the fragment resulted from mutagenic primer, and I am in the condition where I can't change my restriction enzyme. Do the mutagenic primer has some affection to my PCR result bands? Thank you.
The mutagenic primer seems to be binding to two different sites in your recombinant plasmid. One of these sites is the correct one and the other most likely an artifact. Compare the sequence of your mutagenic primer closely with that of the recombinant plasmid. That will enable you to pick out the correct band (based on length) of the two that appear.
Hello
I agree with Vicente Mendoza Reyes , also the links here will help in this issues
Good Luck
https://www.idtdna.com/pages/docs/default-source/user-guides-and-protocols/mutagenesis-application-guide.pdf?sfvrsn=11
https://www.researchgate.net/post/Site-directed_mutagenesis4
One of your primers is able to amplify from a region different than the target.
Try changing the sequence either before or after the site of mutation such that specificity increases in your favor. Alternatively generate the length of DNA in two segments with enough overlap in segments separately, and stitch together in the last step to generate full length DNA.
"I have increasing the annealing temperature and increasing the MgSO4 volume but it did not work."
Try reducing Mg, not increasing.
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