Hi. I performed gradient PCR reaction using a bacterial genomic DNA as template. The annealing temperature of my forward and reverse primers are 59.4 and 61.8 degree celsius. I chose a range from 55-65. But I got multiple bands for each temperature set (a total of 6) and all were faint. I kept 1.25uM working concentration of both primers and 1 ul of template for each set (104ng/ul). Please suggest me what am i doing wrong here and what steps should be taken to get a single intact band?
What size product are you expecting? I don't see a ladder so it's hard to know how big/small those bands are.
Do you have a good positive control you could include?
I agree that without more detail it is hard to advise but assuming that the temperature goes from low to high left to right and that the large bands are not the ones that you want then the third or fourth temperature has got rid of the large bands so this is probably the temperature to use. There is the smallest band which is probably 2 primers annealing together to form a primer dimer. You cannot get get rid of this band by gradient pcr but if you use much less primer ( try half and quarter the amount of primer) and use a hot start enzyme this often gets rid of primer dimer. If you run the gel at a lower voltage then there will be less spreading of the amplified band
Hi all. I was finally able to get a single PCR band. Thank you for all your suggestions.