I am currently working on a research project for my masters using adult Calliphoridae insects. I am trying to examine the COI region but I haven't been successful with my PCR. I'm getting a good amount of DNA (between 300-500 ng/ul) and have samples with decent A260/A280 ratios (like around 1.97) but upon gel electrophoresis I am getting no bands. I am at a lost right now as to what to do to fix this.
PS. I am using the GoTaq Hot start polyermase system.
Hi Jody,
do you think this issue may depend on the primers you are using?
Are there previous works using the same primers on the same species? If not, the issue may be related to the primers.
Possible options would be to try different primers for the same gene or to try to amplify different genes. I would check the literature to see if they suggest any specific primer set and/or if someone else noticed issues amplifying COI. If you find different primers in the literature, try them. Otherwise you could amplify any other gene, (or try the same PCR kit on a different species) just to understand if the problem has something to do with your PCR kit instead (if you can amplify anything else, then the kit is ok).
A last resource, in case there are known issues with the primers for these species, you could try to design your own set of primers by downloading from GenBank any similar COI sequence and check where are the conserved regions..
Hope this helps!
thanks for your info Francesco Martoni . Previous research in my department used the same primers & were successful but those researchers used larvae instead of Adults. so me and my supervisor suspect it’s some inhibitor from adults. I used extracted DNA from larvae as my positive control and I got bands for that.
That's very interesting!
Indeed, it could be some sort of PCR inhibition. By any chance, have you tried to use BSA in the PCR mix? BSA (Bovine Serum Albumine) is known to be often effective. There are other options in case BSA does not work (for example, the T4 gene).
Francesco Martoni I haven’t tried BSA! i’ve Seen it in papers but I’m not sure what concentration I should be adding it.
Hi Jody, I have been using different concentrations and amounts.
To get an idea of what's the best practice, have a look at this paper:
Kreader 1996: Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein.
Good luck!
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