Hello PCR experts,
I am attempting to optimize H3 PCR for my crustaceans, but keep running upon double bands in my PCR product. The attached pictures shows a temperature gradient from 48C to 60C (right to left, top to bottom) for H3 PCR of two of the same species (note that the third well is always the negative control). The brightest band is definitely my H3 region, but, as you can see, one of the samples has double bands, but the other does not. This may have resulted from nonspecific annealing. I am trying to find a way to make my H3 PCR results consistently look more like the second sample (i.e., single banded). I am thinking of maybe shortening my elongation time to reduce the number of long products, but I would love other peoples' feedback first.
I extracted DNA using a HotSHOT method and amplified the H3 region using reaction tube concentrations of 1x GoTaq MM, 0.4uM generic H3aF/aR primers, and sterile ddH2O for a total volume of 25uL with 1uL DNA template. My thermocycler conditions are as follows: (1) 94C for 2min, (2) 30 cycles of denaturing @ 94C for 30s, annealing @ 50C for 30s, extension @ 72C for 1min, (3) final extension @72C for 7min.
Thanks!
I suspect that your 2 samples are a homozygous ( one band only sample) and a SNP or mutation heterozygote within the pcr product. The Het amplifies 4 strands of the same size but as well as the NN and MM bands which run as normal size the final cycle of pcr allows one N and one M strand to re anneal after the denaturation step. The heteroduplex so formed has a bubble in the structure where the N and M sequences cannot base pair at the snp position and the shape of the molecule has changes so it is running as an apparently larger size than the NN or MM species.. Cloning and sequencing would confirm this or even just sequencing if it is a snp. When you get homozygous NN and MM samples them mixing these dnas and amplifying the mixture would produce the same effect. You could also get this effect if there were 2 or more species in your dna which had different sequences but that is for bacteria/virus type samples and probably not relevant to crustaceans
It appears that you have a lot of primer dimer and/or primer excess. Try using a lower primer concentration.
If the lower bands correspond to something else like the loading buffer, then you could also try lowering the Magnesium concentration.
Is your issue solved? Else write to [email protected] or drop a message at www.facebook.com/enzymebiosolutions
Thank you all for your fast responses! This is super helpful advice. I will try lowering the primer concentration and let you know if anything changes. I will also take the mutant heterozygous idea into consideration - never thought of that!
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