Immunoglobulin PCR?

Greg Denomme denomme at FHS.CSU.MCMASTER.CA
Fri Oct 22 23:59:51 EST 1993



On 20 Oct 1993, Fourie Joubert wrote:

> Hi
> 
> I'm having some trouble amplifying hybridoma Fv genes, and it is really 
> starting to drive me nuts!!!
> 
> I started out by isolating RNA from hybridomas, synthesizing cDNA and 
> doing PCR and it worked great. When some of the reagents got used up, I 
> made up a new batch and now nothing works any more ....
> 
> I use Chomckzynski's method for RNA isolation, and the AMV protocol in 
> Maniatis for cDNA synthesis. The problem seems to be in there somewhere,
> because I still succeed in re-amplifying some of my old PCR product, but
> it doesn't work from cDNA. 
> 
> Does anyone know of reagents in the above protocols that I should be
> especially careful about?
> 
> Any help would be enormously appreciated!
> __________________________________________________________________________
> 
>      _/_/_/_/  _/_/_/_/_/  Fourie Joubert           
>     _/            _/     Department of Biochemistry
>    _/            _/    University of Pretoria
>   _/_/_/_/      _/   South Africa
>  _/            _/  bio1 at navi.up.ac.za
> _/      _/_/_/_/                                 Disclaimer: "Fourie who?"
> __________________________________________________________________________
> 

  The method for RNA isolation by Chomckzynski calls for the precipitated RNA
to be resuspended in 0.1% SDS-DEPC water.  When you made up new reagents,
maybe you added the SDS to your DEPC water this time.  That would mess up
reverse transcription whereas older samples would still work in PCR.


Cheers,
Greg
denomme at FHS.McMaster.ca






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