RT-PCR doesn't work with all RNA used - (Aug/25/2011 )
Hi,
I've got a problem with my RT-PCR, an ordinary RT-PCR performed for Actin amplification.
I start the procedure with RNA isolation from plant (Phenol isolation, LiCl precipitation) and I receive a huge amount of RNA by doing that. For cDNA synthesis, I use an oligo-dt-primer.
The PCR itself works -I receive a clear band for the positive control, and I receive also bands with 50 % of the RNA used. But some of the templates show only very weak bands. I diluted the cDNA already because of the great amount of RNA, but it didn' help at all.
Does anybody has an idea about that?
Thanks in advance.
A
-Anneke-
Anneke on Thu Aug 25 09:36:30 2011 said:
Hi,
I've got a problem with my RT-PCR, an ordinary RT-PCR performed for Actin amplification.
I start the procedure with RNA isolation from plant (Phenol isolation, LiCl precipitation) and I receive a huge amount of RNA by doing that. For cDNA synthesis, I use an oligo-dt-primer.
The PCR itself works -I receive a clear band for the positive control, and I receive also bands with 50 % of the RNA used. But some of the templates show only very weak bands. I diluted the cDNA already because of the great amount of RNA, but it didn' help at all.
Does anybody has an idea about that?
Thanks in advance.
A
-nightingale-