Identification of weakly expressed novel gene - (Aug/15/2008 )

Hi all,

Does anybody have any idea how to amplify the low level expressed novel gene? If I cannot get amplicon from RT-PCR, can I use that product as template for nested PCR? If yes, how should I do?

Thanks in advance..

Hi
You can use nested PCR for amplifying the lowly expressed gene. I suggest you gel purify the PCR product of the first round of PCR, and gel purify only the region of interest ( Eg . If you are expecting a 1.5 kb product, I suggest you cut out the portion from 1.0 to 2.0 kb, gel purify the same, and use it as template for the next round of PCR).
Suggest not to keep more than 30 cycles for each PCR.

Regards
Avinash
http://aviprem.gq.nu