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Slippage of PCR polymerame


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6 replies to this topic

#1 XCI

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Posted 08 June 2009 - 11:29 PM

Im 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???

#2 Michaelro

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Posted 09 June 2009 - 10:33 AM

Im 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!

#3 phage434

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Posted 09 June 2009 - 11:51 AM

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.

#4 XCI

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Posted 12 June 2009 - 02:32 AM

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?

#5 perneseblue

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Posted 12 June 2009 - 03:38 AM

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.
May your PCR products be long, your protocols short and your boss on holiday

#6 phage434

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Posted 12 June 2009 - 07:10 AM

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.

#7 XCI

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Posted 15 June 2009 - 03:17 AM

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




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