John R. McQuiston
zje8 at cdc.gov
Wed Oct 13 07:27:46 EST 1999
It could also be primer dimer formation which raising the annealing temp
should take care of. 500bp is a little high for that but it's a possibility.
In article <3803BF8F.5768 at le.ac.uk>, AFS7 at le.ac.uk says...
>Mark Edward Bowen wrote:
>> My PCR reactions have suddenly become dominated by two well defined and
>> reproduceable small MW (~500bp) products. It happens with different
>> buffers, enzymes, primers and templates. The products always appear to
>> be the same size. I can anneal at temperatures up to 72 C and only lose
>> my desired product. I've tried hot start, touch down, Mg and
>> temperature titrations as well as varying template and primer
>> concentration. I'm at a loss. Any ideas? I use a Stratagene
>Do the extra bands occur in the PCR negative control (and RT negative
>control, if applicable)?
>Have you done the following controls:
>1. No template
>2. No primers
>3. No template or primers
>It would also be helpful to give an idea of what you are trying to PCR
>(ie, what organism, what the template is, what primers you have tried.)
>As general points, have you tried the following:
>Decontaminating your pipettors/using new pipettors.
>UV irradiating your PCR master mix before adding the template and
>Using a different water source.
>> Mark Bowen
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