amplification of 5' end of cDNA

I. Hussain BMBIH at leeds.ac.uk
Tue Jul 15 07:27:46 EST 1997


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.

Ishrut
bmbih at south-01.novell.leeds.ac.uk


 



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