RT-PCR - (May/10/2001 )
I am trying to clone the rat GATA-3 gene from total RNA from T cells. I isolate the RNA using standard TRIzol protocol and the A260/280 is 1.8. However I get no PCR products. Anyone can help?
Try to do the RT-reaction with Oligo dT Primers. I already got a view genes this way, which I didn`t get with the specific Primers.Good luck!
There are several reasons that you can not view the products. one is the Bad RNA. this is not the case in your workbecause of the method you use for RNA extraction. next is the copy number of the Gene Transcripts in you RNA preparation. this is important when you want to see yourproduct at the first round of PCR amplification. sometimes the second roung of PCR(using diluted first Round PCR Reaction as Templet) is required to get your product at a aboundant level. Also you can try to use Clontech RT for PCR Kit. The DNA Taq enzyme at this kit is very sensitive. you will be worried about too much product exhibited after the improper RT-PCR condition performed when you use this kit.Anyway, you can get it if there it is.