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How to label a probe for Northern blotting


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5 replies to this topic

#1 vetticus3

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Posted 03 November 2004 - 04:21 PM

Hi,
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?
I'll love you forever and ever.
Vetticus.

#2 caro

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Posted 04 November 2004 - 01:06 AM

Hi Vetticus
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.
Regards Caro

#3 caro

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Posted 04 November 2004 - 01:09 AM

Just found the labeling protocol: StripEZ-DNA from ambion too. You can see the protocol here; http://www.ambion.co...rot/fm_1470.pdf

#4 haui

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Posted 04 November 2004 - 02:22 AM

Hi,

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.

Good Luck


Haui

#5 parasuis

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Posted 04 November 2004 - 02:30 AM

Basically, you make a labeled copy of a given template by:

- 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.

HTH :unsure:

#6 vetticus3

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Posted 04 November 2004 - 04:56 PM

THANK YOU!




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