I have been trying to get my PCR to work and amplify the correct region but can not figure out what is going on with it. I seemed to have nailed it down to something with the primers. One of the primers is fine, but the one that I need to use seems to hinder amplification of the amplicon and create a brighter band below the amplicon and slightly above where I expect primer dimers. I have tried adjusting template and primer concentration but it has not seemed to help too much. The results are attached below. I am having trouble with the primers labelled V1-V3.
I expect that the primer sets are reacting with each other to give a primer dimer which shows as a small diffuse band of 50-100bp. Try running all of your primers together and as mixed pairs using water instead of dna and see if primer dimer is a problem. you could run 1f-1r, 1f-2r.1f-2f , 1r-2f and 2f-2r as well as the full set of 1f,1r,2f and 2r and it should show you where the poorly matched primers are. You can also (to solve the problem) use half the amount of all primers and use a hot start polymerase
Which band sizes do you expect for each primer? What is your amplification temp. And duration?
Could you share some more details on topics such as thermal and pipetting protocols?
Try reducing volume of primers, the troubleshooting when it comes to primers might sometimes reside in the smallest of details that we wouldn't consider.
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