Are these primer dimers?
hopkinsc at lincoln.ac.nz
Thu Feb 13 10:28:32 EST 1997
Sorry to follow up on my own post, but it has been pointed out to me that I
need to give PCR conditions before an answer can be given.
We are using Taq polymerase (1U/25ul reaction), 0.1mM dNTP, either 2uM or 10uM
of primers (it depends on the reaction, but the primers are always in equal
concentrations in the PCR), 0.8ng DNA, a total of 2mM Mg2+ and 33 cycles.
These conditions haven't changed, but we have only been getting these bands
for about 3 weeks.
The main question is can both these bands be primer dimers (as they do have
that look about them), and if not what is the second band?
>> We have a postgrad student who is starting to have problems with PCR that has
>> got us confused. It is looking like she has two sets of primer dimers her
>> gels. Both bands occur under 100bp in length,and it is clear that they are
>> two distinct bands (I would suspect about 50 and 90 bp - our ladder
>> below 100bp so I can't be more specific). We suspected the top one to be
>> contamination, as it also occurs in the negative, so tried all new chemicals,
>> but they are still there. It doesn't always occur in every tube either
>> (perhaps 3 out of the 5) and there is no correlation with individuals or
>> species. We have tried a different DNA region (i.e completely different
>> primers) and they still occur.
>> This morning she went to rerun samples that were 5 days old and her expected
>> band had completely disappeared but she still had these two blobs at the
>> bottom. We then thought DNAses could be contaminating the samples while they
>> are in the fridge, however she has just taken sample straight out of the PCR
>> machine and have run them on a gel and the bands are present. Could it be
>> DNAses or should the PCR destroy them? If this problem is being caused by
>> DNAses why doesn't it happen in all tubes as a master mix of all but DNA is
>> made up each time?
>> Has anyone experience this or can explain what is likely to be causing it?
>> Many thanks
>> Charlotte Cameron email: hopkinsc at lincoln.ac.nz
>> Lincoln University
>> New Zealand
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