Slippage of PCR polymerame - How do I avoid it ? (Jun/09/2009 )

I´m doing some kvantitative analysis of DNA fragments amplified by PCR using a AmpliTaq polymerase.
The analysis is done on a ABI machine but do to a 43 x TC repeat several shadow bands are produced, which make it difficult to kvantify the product.
How can I avoid this??? Is there another polymerase I can use in stead or can I change the PCR programe???

XCI on Jun 9 2009, 10:29 AM said:

You may try to change number of parameters
The first one is annealing temperature. Higher annealing temperature may reduce the amount of non-specific bands
Another parameter is magnesium ions concentration. Try to reduce/increase the magnesium in your reaction.
Changing the polymerase may also be a solution.
Good luck!

PCR is not the only DNA replication process that introduces slippage. You may find that your template DNA also has variable numbers of repeats.

I would try lowering the extension temperature to 60-65.

phage434 on Jun 9 2009, 09:51 PM said:

It is actually the variable number of repeats in the DNA that I am interested in investigating.
I tried lowering the extension temperature to 65 C and also lowering the number of cycles, but it didn't make any difference.
Any other surgestions?

try using a proof reading DNA polymerase.

I would use Phusion. Since this DNA polymerase is fused to a dsDNA binding protein, it should not experience as much slippage.

I'd second the Phusion suggestion. But why do you think that the product you are seeing is not an accurate representation of the template(s) present in your template DNA.

phage434 on Jun 12 2009, 05:10 PM said:

I am using the area under the curves ( representing the peaks ) for quantification. This is why I am only interessed in one peak for every template DNA