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hezma 07-23-2008 11:53 AM

cDNA Synthesis Problem
 
Hello i am having problems synthesizing full length cDNAs. what could be the problem? I am extracting RNA from cells then using Reverse transcriptase to get the RNA to cDNA however when i clone the cDNA into plasmids, the ones i am looking for are not there or short.

plz help

danfive 07-23-2008 07:49 PM

Re: cDNA Synthesis Problem
 
Improve the quality of your RNA and the problem should go away.
I use Trizol, but a good kit can give you even better results. Also try to use fresh RNA since it will degrade, even if frozen.

vimlendusinha 07-29-2008 12:41 PM

Re: cDNA Synthesis Problem
 
just use GTC or any kit for rna isolation and do the RT. check ur cdna synthesis protocol u have to denature rna with oligo dt, cool it and then add dntp, buffer and enzyme and keep at 42 for 1hr u wil get it. The quality of rna should be good

zulcyq 10-26-2008 01:29 AM

Re: cDNA Synthesis Problem
 
hi

try to use omniscript for 1st strand synthesis which helping in reducing the production of short 1st strand by normal RT. then, during PCR, use hot start PCR. should be better result and can eliminate non-product band

indri 06-19-2009 09:23 AM

Re: cDNA Synthesis Problem
 
while extracting mRNA, always keep the tubes (with mRNA pellet inside) on ice to prevent degradation of mRNA by RNAse. Just a tip.


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