Good day! I extracted bacterial DNA from (rotted) apple fruit samples and am planning to run a 2-step PCR. This result was from the 1st PCR. This was from non-diluted DNA samples of about (20 - 50 ng/ul), I used primers with overhang attached, and used DMSO for the reaction.
I have previously tried (not shown) to dilute these samples but the target DNA just keeps getting low and the 2nd band is not eliminated. I am at a lost now how to troubleshoot this problem. Will MgCl help? Any and all advise is highly appreciated. Thank you!
Rocel Amor Indong Hi!,
you can try setting up a gradient to check for optimum Annealing temperature, probably it can help you.
Chriswin Saimon Hi! I tried your suggestion and adjusted annealing temperature from 55 to 60, increments of 2, but I still have the same problem with the 2nd band. I will try to increase temperature up to 65 but if you have any other suggestion I can try, please let me know. Thank you!
Can you please share a picture of the agarose gel for the PCR products?
So you can see here I lost 2 samples (D1 & E2), I got bands from those at 55deg annealing temperature. And the rest is just the same as the previous gel. Chriswin Saimon
Rocel Amor Indong Hi!, the bands visible on the gel, are they matching the amplicon size that you are expecting?
one suspicion I think is, that the above bands are your products, whereas the bands below might be primer dimers. Can you try to reduce the volume of the primers and check once?
This is all I could think of now.