help PCR

[Tom]

jkennie at GPU.SRV.UALBERTA.CA (J Kennie) wrote:

>On 1 Oct 1997, 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
>> 
>> 
>Julian,

>I have been having the same problem. My template should be fine, the
>primers I use are kept at -20, I've tried new buffers, enzyme, water,
>dNTPs, etc. and I still get smears in all the lanes. Incidentally, this
>started happening a few days before I went on holiday and is still a
>problem now that I'm back. 
>Does anyone have any suggestions on how to fix this problem??
>I am amplifying a 850 bp fragment using primers that are about 30 nt long.
>I have previously had it working fine. 
>Help.

>Jan 
>University of Alberta

Clearly the message here is that people doing PCR shouldn't go on
holidays ever

Tom