Are these primer dimers?

Hopkins, Charlotte 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?

Charlotte

>> 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
>doesn't go 
>> 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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