help PCR

Anita Malhotra a.malhotra at bangor.ac.uk
Mon Oct 6 11:12:59 EST 1997


Mario Chin <user at grgs.com> wrote:
>
>
>Julian Robinson 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
>
>
>If the -ve control also have smear like the other lanes, the template is
>probably degraded.
>--
>de mario
>

A negative control has no template in it, so this is the one thing it 
can't be due to. I normally don't waste too much time trying to work out 
exactly what's wrong, I just make up new reagents and start again. 
Sometimes works (but I note that you have already tried this). Other times 
is seems to be due to the phase of the moon or not praying to the right 
gods (i.e. who knows). However, a change in the weather does seem to have 
some correlation with things suddenly going wrong. Sorry, not a very 
useful reply.

Anita Malhotra
School of Biologial Sciences
Unviersity of Wales, Bangor




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