RT-PCR by using in vitro synthesis RNA

Jing Zhang jzhang at sfu.ca
Wed Oct 14 14:11:30 EST 1998


Dear Netters:

thanks for your responses to my questions about DNA gel purification, it's
really helpful.

Right now I did RT-PCR by using in vitro synthesized RNA, but I found the
signal of RT-PCR is very weak, and it's weird PCR without RT can get even
stronger bands than RT-PCR which means DNA contamination. have you ever
noticed MuLV reverse transcriptase could inhibit Taq in RT-PCR.

 In vitro synthesized RNA should  be 100% pure specific RNA, because (1) I
use specific DNA template which on the downstream of T7 promoter. (2) I use
PCR downstream primer to synthesize cDNA before doing PCR. It is believed
RT-PCR of in vitro RNA should get very strong signal, even diluted x1000,
still should have singal after PCR.
any experience or suggestion will be really appreciated !!

JING ZHANG





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