help PCR

David MacHugh dmachugh at mail.tcd.ie
Wed Oct 1 13:40:32 EST 1997


In article <833B3E5B4C at trans.plants.ox.ac.uk>,
julian.robinson at plant-sciences.oxford.ac.uk wrote:

>       Iam trying to amplify a ~450bp fragment with two 20bp primers. 
> It was working perfectly before I went on holiday. However when I 
> came back I begtan to have problems. The problem was smears in all 
> the lanes, including the control lane. It wasn't the template DNA 
> that was the problem (it oocured in the -ve control), and it is'nt 
> contamination (new everything has been used). 
> We are leaning towards primer dimers as the answer at the moment (as 
> there is some complementarity? between the primers), but the real 
> question is why has it just started to occur, when it was perfect 
> (i.e. no smears and clean bright product) before I went on holiday?
> 
> many thanks 
> julian
> ---
> julian.robinson at plant-sciences.oxford.ac.uk


{I'm not sure whether you tried a new batch of primers, but the following
may help}

I had a similar problem with primers I was using to amplify archaeological
DNA.  The PCRs were working beautifully for a number of weeks after I
received the newly synthesised primers.  Then suddenly the PCRs became
horrible - the products if present were very faint.

I presented my problem to this newsgroup and got some very useful
comments.  Seemingly, primers are actually quite labile and can "go-off"
very easily, particularly if the amplifications are from difficult
templates.

The solution to this is to store the primers in aliquots at -20C (I use
ten aliquots for each primer) and keep a working aliquot in the fridge for
no more than a few weeks.  This ensures that the primers are not
continually being freeze-thawed.

If you re-order your primers and store them like this, I'll bet you will
not have this problem again.

A good article which discusses this topic is:

P.N. Hengen, Wayward PCR Primers, Trends in Biochemical Sciences 20: Jan.
1995, pp. 42-44.


Hope this helps.

David.


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