amplification of 5' end of cDNA

John Watson watson_j at bms.com
Thu Jul 24 16:10:18 EST 1997


I. Hussain wrote:
> 
> I have been trying to amplify the 5' end of cDNA by RT-PCR followed by dG
> tailing using terminal deoxynucleotidyl transferase to add on a stretch of
> G's to the 3' end of cDNA following first strand cDNA syntheis.I am then
> using a dC primer and a specific reverse primer to amplify the 5' end. I have
> had no success.
> I am using the following tailing method:first strand cDNA + 1ul 6mM GTP + 15U
> terminal deoxynucleotidyl transferase at 37oC for ~30min. Reaction terminated
> by heating at 70oC for 10min.An aliquot of this is then used for second
> strand PCR.
> I don't know if my tailing procedure is working?
> Does anyone have experience of successful dG tailing and/or can recommend a
> procedure?
> I'd be grateful for any tips/ suggestions as to where I'm going wrong.

We routinely use the Gibco-BRL 5' RACE kit. It has worked in most cases
(the jury is still out for the most recent experiment). It you can
afford it, you might give the kit a try.

AJW
----------------
John Watson
Bristol-Myers Squibb Co.
watson_j at bms.com
---------------------------------------------------------------------
"If you're not part of the solution, you're part of the precipitate."
---------------------------------------------------------------------



More information about the Methods mailing list