tom at senet.com.au
Wed Oct 8 17:39:44 EST 1997
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.robinson at plant-sciences.oxford.ac.uk
>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.
>University of Alberta
Clearly the message here is that people doing PCR shouldn't go on
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