6 replies to this topic

[XCI]

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???

[Michaelro]

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

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???

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!

[phage434]

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.

[XCI]

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

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.

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?

[perneseblue]

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.

[phage434]

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.

[XCI]

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

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.

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