primer extension anyone ?

David L. Haviland, Ph.D. haviland at KIDS.WUSTL.EDU
Mon Feb 5 18:45:53 EST 1996


On Mon, 5 Feb 1996, A. Kumar wrote:

> Hi ! I have been trying primer extension for a long time now and it has 
> never worked. I am using a 28mer oligonucleotide as a primer. It labels 
> well and I get a high specific activity. But it just doesn't seem to 
> hybridize to the RNA. I have checked the sequence and it is correct. I 
> have been using total RNA. Should I use mRNA ( poly A ), or maybe try and 
> purify the RNA ? Also, if anyone could tell me a source for a good and 
> reliable protocol for primer extension, I shall be grateful. Please help! 
> Thanks.
> Ashish Kumar

Ashish:

I used the "Harvard Manual" for the basic primer extension protocol.  For
my primer extensions, I used oligos of no less than 40 bases in length so
that the hybridization would be optimal.  Make sure the oligo reads
opposite to the reading frame of the message - make sure it's pointing
upstream.  The type of RNA used depends greatly upon its abundance in the
tissues where you isolated it.  In my case, I was looking for a low level
complement component and needed 3 ugs of Poly A+ mRNA or about 30-40 ugs
of total.  Both worked equally well.  Do not forget the tRNA control. 

Hope this helps,
David

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