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PCR product wrong size (too high) - PCR (Mar/17/2005 )

Hi everybody,

I have a problem with my PCR ... I am getting a very clear product of 350kb instead of 218 kb...I tried different primers that are in between the target sequence (siRNA)... the same wrong product appears for all of them...
I tried to desalt the primer...no changes.
When I try to design other primers with a bigger flexibility in the primers surrounding the target sequences (ex: 920 position of the target, 50 bases surrounding the target), I get the same bench of primers...
When I do the blast to check the primer specificity, I only get the wanted gene related to these primers...
I use: one PCR cycle consisted of the following: 94C for 1 min, 60C for 1 min 30s and 72C for 1 min 50s

Does anybody have some suggestions?

Thanks in advance,

Francoise.

-Francoise-

Hi Francoise,
Are you trying to amplify cDNA from RTrxn? Should an insertion be an explanation?good luck cool.gif

Ribosoul

-Ribosoul-

QUOTE (Ribosoul @ Mar 18 2005, 03:02 PM)
Hi Francoise,
Are you trying to amplify cDNA from RTrxn? Should an insertion be an explanation?good luck cool.gif

Ribosoul

Thanks for your answer.
Well, I am trying to transfect some eucaroytic cells with siRNA to inhibit a gene expression; I need to use RT-PCR to verify that this gene is effectively inhibited.

-Francoise-