How to label a probe for Northern blotting
Posted 03 November 2004 - 04:21 PM
I'm going to be starting a northern blot, and I've been reading up on, I got a protocol, all good, but I still don't really get how to label a probe. Is there anyone out there who would be willing to enlighten me... please?
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Posted 04 November 2004 - 01:06 AM
I have labeled the probe with [alfa-32P] ATP with exonuclease free klenow, before hybridising.
I have used NothernMax from Ambion, it is really good.
Posted 04 November 2004 - 02:22 AM
I agree with Caro. I also use the Ambion Strip EZ DNA random prime labelling and also Strip EZ RNA (T7,T3 RNA Polymerase) Riboprobe(RNA gene sonde).
Both are working good. For random prime we take 32P dATP.
The big advantage with this kit is you can often strip the membranes and can use for many hybridisations (10-20 times). Because of this special modified dCTP.
Posted 04 November 2004 - 02:30 AM
- denaturing a double-stranded template
- adding random hexamer nucleotides as primers
- adding one labeled nucleotide (DIG, or radioactive, alpha-P32-dATP or-dCTP)
- adding the other three nucleotides in unlabeled form (!!)
- adding Klenow fragment
(plus buffer etc)
Some of the random nucleotides will find a place in which to bind to your template, and the Klenow will start to make a copy of the template strand from there, incorporating the nucleotides including the labeled one, which will result in a labeled copy.
Before hybridization, you'll need to denature again because the labeled copy is still attached to the non-labeled template strand.