PCR anomaly

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.

John







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
>> robocycler.
>
>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
>primers.
>Using a different water source.
>
>> Mark Bowen
>
>love
>Anna




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