Doing a nested PCR for Blastocystis useing primers RD3 and RD5 for the first PCR and primers RD5F and BrRDr for the second PCR. I'm getting bands on the gel but also getting a lot of smearing. Does anyone know how I could resolve this?
A gel image would be useful but I expect that you have great over amplification of yout template and the template is annealing to other template molecules and forming long smears. Every 10 cycles of pcr is 1000 times more product so if you are running 30cycles then 30 cycles your could be amplifying up to 10exp18 amount of dna, Too much. Try diluting the first round pcr product 1:100 and setting up pcr tubes with the nested primers and remove tubes at 12,15,18,20,23 cycles and see which one gives you a strong clean product
Use 1ul of a 1:100 dilution from the first PCR product as a template for the second PCR. Smearing is typical of excessive DNA before amplification.
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