primer extension

[David L. Haviland, Ph.D.]

On Mon, 1 Apr 1996, EFDG wrote:

> Does anybody know of a protocol which labels primer extension products by 
> incorpration of alpha-labelled 32P instead of end labelling the primer with 
> gamma-labelled 32P dATP?

Stuart:

Been there.  I had the very same thought when I was doing primer 
extentsions and all I did was take the protocol in the Harvard manual and 
swap the same concentrations of dNTPs with dNTPs-dCTP.  All I obtained 
was an enourmous mess of an autorad - i.e., didn't work.  I was looking 
at a relatively low abundance message which was why I tried modifiying 
the protocol to begin with.  I gave it another couple of attempts all to 
no avail.  The only way I managed to get them clean was to spin the end 
labeled oligo over a G-25 column where there was sizable separation from 
the labeled oligo and free 32P.  In my case, I used the BMB g-25 spin 
columns.  OH.. btw- my oligos for primer extenstion were no less that 
37-mers.

Hope this helps,
David

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