multiple bands in pcr product
how to remove multiple bands from pcr product
let me know if your positive control DNA template and source is the same as others'... as seen in the gel image it is quite ok unlike others..
PCR is sometimes crazy but if every reagent in reaction is in its optimized concentration and also the temperature and primers so it is hopeful to get desired result.
some times increasing the annealing temperature may help to solve such a problem. however not to forget about enzyme and MgCl2 optimum concentration.
and the last but not the least is your primers; it is very important to have well designed primers so that to have a good amplification.
All very good suggestions by Saber. Like Saber wrote, I would begin with raising the annealing temperature and move from there.
Thank you very much for your suggestions! I will do it. My positive control (PC) was a vaccine virus, the samples are chicken thymus (checking for Chicken anemia virus). It's always these samples that give me trouble, the other tests for A.I. and Adeno are never problematic.
Good day, I increased the annealing temp to 62C from 60C (from the journal I am using). However, bands were still visible. Usually, how much increase is ideal? Also, I use KOD Dash for my PCR instead of the usual Taq polymerase. The manual states that KOD Dash has 1.2mM MgCL2 in the final mix. Do you think this is my problem? I am thinking about using 2x or 3x the normal amount of the KOD Dash to compensate. I think there really is something wrong in my technique, even the Negative control is now showing bands (although I do not have this problem with A.I. and Adeno virus).
It is better than before, though unfortunately, you negative control lane is lighting up. Judging from your markers (though unlabeled) I guess you are trying to amplify something between 500 - 2kb? The KOD Dash (a mix of regular and proof reading polymerase) appears to be an overkill for this kind of amplification. What is the calculated Tm of your primers?
As far as negative control lighting up, please discard whatever PCR grade water (hopefully in small aliquots) you are using. Switch to another set of pipettes and assemble PCR reaction in a place where there is less or no human traffic.
Dr. Ali, my target is 186-bp and my markers are 100-bp. The Tm of my primers are: 61.2 (CAV-1) and 61.7 (CAV-2). Aside from KOD Dash, I have sigma Taq polymerase, would you suggest it in this case? Thank you very much for your time.
Hello Fletcher,
Please try Sigma Taq. Please do a few more things:
1. Setup reaction on ice.
2. Add Taq polymerase when tubes were hot in the PCR machine (hot start the poor man's way).
These two measures may help with reducing if not eliminating non-specific bands.
Your annealing temp is already high. You may want to increase it by another 2 Celsius.
BTW, have you written to the author of the paper that describes the use of these primers? They could help you with PCR optimization.
Regards.
Dr. Ali, thank you very much for your advice. I will do it. I really appreciate your help!
Yes also I think 62c is better than 60c... would suggest to run a gradient PCR with annealing tmp ranging 62 to 67 (62, 63, 64, 65,66,67c). I think MgCl2 concentration is ok not affecting your problem. first increase the annealing tmp if it is not working then consider the extension time, number of cycles, primer concentration, template DNA concentration by reducing their amount.
about the enzyme, would not suggest to increase its concentration.
and about your negative control; as you didnt have such problem before and also based on your positive control, I think that s not a big issue and may be caused by pipetting or some where else, do it again if facing again then think about it as an issue ;)
Either you increase the annealing temp. or you cut out the band and re amplify to see whether you got the specific band or not
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