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Weird bands in standard PCR of gDNA and cDNA


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#1 Montys

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Posted 15 March 2011 - 05:01 AM

Hi,

I have a question regarding PCR of gDNA and cDNA.

We are looking at a CDS of a gen. This CDS is only in one exon located. We have primer that span this CDS. When we now do PCR of gDNA we get perfect bands of the specific fragments of the CDS region. When we use cDNA it gets odd.

I get PCR products of the shorter fragments of the CDS but non of the longer fragments. I would say it is a truncated mRNA we have. Additionally I get for the largest working fragment TWO bands in a gel but only for the cDNA not for gDNA, here I just have one band.

When I use the commercially available GoTaq-Green Mastermix I just got my "real" one band also in the cDNA. The second band just appears when I use my standard Taq. I blasted of course all primers against the hole genome and additionally against the Gen, with no significances except for my gen of Interest.

Does anybody got any idea? First I thought ok new splice variant the one Exon are actualy two exons with one Intron or a truncated cDNA of unknown reason , but then with the GoTaq no second band. How comes the difference between gDNA and cDNA.

Thx for yours help.

Andreas

#2 Trof

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Posted 16 March 2011 - 12:24 AM

If you get two bands with common Taq and single one with a "better" mix, it's more probable that you only saw nonspecific ampification. Do you pipette your Taq reaction on ice or use a manual hotstart?

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#3 Montys

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Posted 17 March 2011 - 12:44 AM

If you get two bands with common Taq and single one with a "better" mix, it's more probable that you only saw nonspecific ampification. Do you pipette your Taq reaction on ice or use a manual hotstart?


We do it on ice. But why I don't get the second band in the genomic DNA just in the cDNA. If it is unspecific one would see the bands in either the gDNA and the cDNA.
But you are right why I don't get the bands with the commercially available Taq?

Anybody another good idea?

thx




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