Please kindly send -Protocol for labeling PCR primer to
use for Northern probe.
Hiranya S. Roychowdhury
hroychow at NMSU.EDU
Tue Dec 8 17:36:16 EST 1998
At 08:04 AM 12/8/98 -0800, G.E. Woodward wrote:
> Could you kindly e-mail me a protocol for labeling a PCR primer
>to use to probe a Northern blot and also a protocol for the proper
>hybridization and washing conditions. Many thanks.
> Yours sincerely,
> Geoffrey E. Woodard
Have you checked in any Mol Bio manual (eg. "the Red Book", Maniatis etc.)?
You can end label the primers with 32P (from gamma-32P-ATP) using T4
polynucleotide kinase. If the primers are not phophorylated, proceed with
the same protocol as you would while phosphorylating it, substituting cold
ATP with hot. If it is already phosphorylated, then do an exchange reaction
using the same substrates.
As for hybridization and subsequent manipulations, the conditions should
depend on the complexity of the target, the probe, homology expected etc.
But you may try the Church & Gilbert method which works really well for me
and for others that use it.
Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu
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