Hello,
I'm trying to amplify a gene in both eDNA and bacterial DNA (a strain of Bacillus, that should be the positive control) using degenerative primers, their sequence was published in a (decent) paper.
However, following the exact authors protocol (with the exception of the buffer used)- I'm not getting any bands whatsoever.
I've been trying to do gradient PCR- but still, no bands (67.5 is the authors recommended annealing temperture, I've tried the whole range of 56-72). I found out that the buffer I'm using (Dream Taq) has 20 mM of MgCl, while the buffer that was used in the paper (FailSafe PCR Buffer) has 4 mM of MgCl- could that be the reason?
I currently don't have a positive control, by using the same reagents but dufferent primers, and the same PCR machine yielded efficient amplification, so I believe I'm missing something.
Thansk you.
Time to start trouble-shooting to find a good positive control. You've learned that your enzyme works, your thermocycler works and you can set up a successful PCR reaction. That's a good start! Try out a different set of primers with your Bacillus DNA to confirm that your DNA extraction worked. Then test out the published primers. I wouldn't think the MgCl matters that much. What are the recommended annealing and extension times?
Thank you for your answers.
The annealing recommended time is 30 seconds and extension is 45 seconds, with final extension of 5 minutes.
I do have another set of primers for that gene cluster, but the one I'm using right now is quite universal therefore I preferred to use it.
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