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PCR problem: DISTINCT bands far shorter than target!


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#1 lookinglassgirl

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Posted 26 February 2005 - 01:26 PM

We are having a rather odd PCR problem. I am cloning a gene from a cDNA library. When we run the gel of the PCR reaction, I am getting one very strong and distinct band at about 700bp, a less strong but still distinct band at 500bp. There is pretty much no smearing on the gel, and the gene is supposed to be about 2500bp. I have looked at troubleshooting sites but none of them describe this problem, and there is no genomic DNA for this gene yet, so I have to make this work.

Here's my rxn. conditions:

1ul template (cDNA lib)
10ul NEB thermpol buffer
10ul of each primer @5uM
3 ul dNTP's @ 10 mM
1ul Vent pol.
water to 100ul total volume

I am new to PCR and any help would be awesome!!!

#2 Panoramix

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Posted 26 February 2005 - 04:09 PM

To help you we need to know also the concentration of the Mg2+ (I suppose it is included in the reaction buffer) and the conditions in the cycler (temperatures and times you used) !

I had that problem also with a big (~2kb) product and addition of 5% DMSO solved the problem. You could test several DMSO concentrations as you have to add the less needed as DMSO increases the rate of mistakes !

Good luck !

Edited by Panoramix, 26 February 2005 - 04:12 PM.


#3 lookinglassgirl

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Posted 27 February 2005 - 08:59 AM

The NEB buffer is: 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl, 2 mM MgSO4, 0.1% Triton-X-100, pH 8.8 @ 25 C. Supplied as a 10X concentrated stock.

I do 38 cycles at: 94.0 (1min), 55.5 (1min), 72.0 (2min)

#4 methylnick

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Posted 27 February 2005 - 06:54 PM

Hello,

I was just wondering, does the gene sequence length include introns because if that is the case, if you are amplifying from a cDNA library, ie: DNA from mRNA transcripts which would be devoid of introns and hence the shorter product from expected gene length.

If you are getting two products from the one primer pair this would possibly mean alturnative splice transcripts from the same gene.

Hope this helps

Nick




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