PCR product dimer issue - (Apr/08/2010 )
If the longer band has same primer sequence as your product it could interfere with the sequencing, it will increase background and it will be more difficult to find a mutation if it's only in a portion of cells, or it would render the sequence unreadable if it's more concentrated. You should try to get only single band to sequence.
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'm going to try that today and we'll see if that helped Thanks!
Was one of those lanes produced by your untagged primers?
In addition of what others had said, I would like to add in my idea.
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.
I can't reduce Mg concentration because it's already in a master mix..... I've tried lots of enzymes and that one which I use now is easy and always work for for those genes which I need to amplify now. Hmmm it used to work giving nice single band when I was using untagged primers. I'll give a try today Adrian, and I hope it'll work as I'm really fed up with that M13 tag :/
I've tried to reduce annealing and extension time. I tried to amplify 2 exons:
A - annealing as normal, extension reduced by 15s
B - annealing as normal, extension reduced by 30s
C - annealing reduced to 15s, extension as normal
D - annealing reduced to 15sl, extension reduced by 30s
Should I keep annealing time reduced to 15s and keep reducing extension time?? But the correct band is getting weaker... Or maybe I shouldn't worry about all of that as it's product dimer so the sequence will be the same but let say longer....
How many cycles of amplification are you doing? Given the strength of the main product you could cut 5 cycles off.
Have you tried touchdown PCR? It might help you swamp out the secondary reaction.
I'd still take a few samples with varying amounts of the larger band and see what the sequencer makes of them.
rudzielec on Apr 14 2010, 08:06 AM said:
How do you know that?
What else could be there if it's always double size of correct PCR product. I've tried to amplify my stuff with normal primers and then I used as a template product of first PCR and amplify that with M13 tagged primers and I still got that....
If it'd be sth like unspecific binding to the template (gDNA) it'd be always the same size doesn't matter which exon is amplifying..I think...
I'll probably give some samples to be sequenced and we'll see if that extra band makes actually any difference. So fingers crossed