PCR product dimer issue
Posted 08 April 2010 - 04:50 AM
Have you got any suggestion what else I can try to get rid of that double size band?
Posted 08 April 2010 - 06:13 AM
How big is your PCR product? Can you adjust your extension time so only the shorter (correct) band appears? Can you use another sequencing primer tag, other than M13?
Posted 08 April 2010 - 03:28 PM
The fact that it's always there, no matter what you change, suggests it might be something unrelated to the amplification reaction. How big is the expected product? The second band (apart from just 2x the expected band)? What template are you using? How was it prepared? Have you changed all of the reagents? Have you cleaned your pipettes / used someone else's pipettes / used someone else's machine? Have you changed PCR preparation rooms?
Most of all, could you upload an image of the gel?
Posted 09 April 2010 - 12:40 AM
PCR products are 200-600bp. When I tried to cut the extension time down previously I was getting my correct band fainter and other unspecific bindings didn't disappeared completely. But haven't tried that for PCR with the M13 tag. The problem is that I can not use other sequencing primer tag I use gDNA as a template. I don't think that the problem is because of pipettes, reagents or machine because other PCRs with normal (without M13 tag) primers work very good. I've uploaded one of my gel images. That double size bands are quite faint but previously when I purified (on a column) PCR products with extra bands as faint as those, I had quite noise sequences in some of them. I don't have a proper software so sometimes it's quite hard to say if it's mutation or not I've got lots of samples so I can't spend lots of time contemplating if that's just noise or maybe mutation or repeating PCR and sequencing again If it'd be possible I'd like to have nice sequencing results
Posted 09 April 2010 - 02:45 AM
I don't understand -- is your M13 tag a 5' extension added to your primer? Can't you just re-synthesize your primer with a different 5' extension?
Posted 09 April 2010 - 02:51 AM
Posted 09 April 2010 - 03:46 PM
You could always try http://www.genewiz.com/
I haven't used them yet, but I've heard it's easy. I suspect there are a lot of companies out there that will sequence your PCR product for you on the cheap.
Posted 11 April 2010 - 11:44 PM
Posted 12 April 2010 - 04:02 AM
It may be that your M13-tagged primer has to be purified by your primer synthesis company using HPLC or gel purification to remove truncated synthesis products, insuring that you only have full-length primers.
You may also have to fool around with your PCR mix (e.g. Mg++ concentration) or your parameters (e.g. number of cycles) to stop amplification of your spurious product or minimize it to a degree that it doesn't interfere with sequencing.
Posted 12 April 2010 - 04:31 AM
Posted 12 April 2010 - 07:06 AM
A have another idea, run a first round of PCR with your untagged primers, you should get a clear single band as you said, then dilute it many times (like 1:10 or 1:100) and amplify with the M13 tagged primers. There should be no non-specifities in that second round. Only drawback may be, that more cycles means higher probability to get an polymerase error, but you can try lowering the number of cycles in the first PCR.
I never trust anything that can't be doubted.
Posted 13 April 2010 - 12:52 AM
Posted 13 April 2010 - 08:25 AM
Posted 13 April 2010 - 10:45 PM
If I were to get rid of the extra "larger" band, I personally would reduce further my annealing and extension time. This will not give "enough time" for Taq to have chance for larger amplification.
Try to reduce annealing time by 5-10seconds, and reduce extension time for 15 seconds. Or, you just use 10-15 seconds for your annealing time, and skip the extension time.
I do agree with Homebrew to reduce MgCl2 concentration, and reduce your buffer strength as well.
You can also try touchdown PCR.
Hope this help. Keep us update.
Edited by adrian kohsf, 13 April 2010 - 10:48 PM.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
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