amplification of 5' end of cDNA

Andrei Popov andrei.popov at SPAM.bbsrc.ac.uk
Tue Jul 15 12:56:43 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.

In my hands oligo dG did not workeither for PCR. Then I changed it by making
a primer like:

5'-NNNNNNNNNNNNGGGGGGGG-3' where NNN.... is random sequence, preferably the
one corresponding to a primer that you have in your freezer and which has
worked before for PCR from another species :-)
 
 
> I'd be grateful for any tips/ suggestions as to where I'm going wrong.
>
> Ishrut
> bmbih at south-01.novell.leeds.ac.uk
> 
> 


about half a year ago I posted here a complete protocol together with
the sequence of the 5' primer which worked for me. Try the bionet.molbio.methds-reagents
archives.
One thing more: you must REMOVE the NNNNNNNNNN.....GGGGGGGG primer
before PCR by running the reaction mixture through G-50 column or by  any
other method.


all the best 


-- 
Andrei Popov

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