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RT-PCR doesn't work with all RNA used

RT-PCR oligo-dt Actin

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#1 Anneke

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Posted 25 August 2011 - 01:36 AM

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

#2 nightingale

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Posted 11 September 2011 - 03:21 AM

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


have u tried using random primers ???
" The more you learn, the more you realize how little you know ... "





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