Mystery PCR product at lower dilutions - (Jan/19/2007 )
Hi,
I was wondering if anyone could help me with a real-time query i had.
I have developed and optimised a real-time SYBR green assay, incorporating my own set of primers which give a PCR product of about 100bp.
When i ran a serial dilution on purified PCR product to get an idea of the sensitivity after about 1fg i got a second smaller PCR product which is not present in any controls.
This only appears after about the 1fg dilution.
The same thing also happens when i insert the PCR product into a plasmid (again only after 1fg dilution)
Does anyone have an idea as to what this could be?
Any guidance would be much appreciated
What's the lenght of the product??
When run in a 3% gel against a 10bp DNA ladder the mystery product appears to be about 70bp in size
(this is purified PCR product)
A band also appears as expected at 100bp i.e. the desired PCR product
If this appear only at a so diluted sample that had been purify the only thing that comes at my mind is that it is just the same sample but had not finish to polimerize.because can't be contamination (it will appear in undiluted sample), primer dimer will have a lower lenght.