PCR anomaly

A.F. Simpson AFS7 at le.ac.uk
Tue Oct 12 18:09:03 EST 1999


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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