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RT-PCR

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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RT-PCR

Postby sisley03 » Thu Dec 23, 2004 12:39 am

I have done RT-PCR to get p16 gene,but till now,
electrophoresis result is always show 100bp,what happen? i used kit,adjust my method, but no matter how i change my methods, the result is still 100bp. What method could i use to get my satisfactory result? Thanks 4 ur help!! :P
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Postby Raegan » Fri Jan 28, 2005 12:52 am

Ok,
have you tested out the primers first to see if they work?
I mean, RT the RNA and then run a normal PCR (preferably on gradient) to see if you get the band size you want.
do your primers go through an intron?
have you tried different temps?
have you tried different Mg++ concentrations?
what concentration of cDNA do you use? to much can inhibit the reaction
what concentration of the primers do you use? to much is very bad, too little is also bad.
You disturb me.
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