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Help to realize northern blot with non radioactive probe

RNA - Northern blot

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#1 kpoupou

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Posted 19 February 2014 - 04:40 AM

I would like to realize a northern blot with  a non radioactiove probe :

 

1- What do you think about biotin-labeled probe?

 

2- Do you use "Licor system"?

 

3- Which percent of unmodified dTTP and biotin-16-dUTP?

 

Thanks for your help!



#2 phage434

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Posted 19 February 2014 - 05:33 AM

I prefer a dig rather than a biotin probe. Roche is the primary (sole?) provider. I'd suggest 10-25% dig or biotin - UTP. Don't forget that you can't use most high fidelity PCR enzymes with dUTP -- Use straight Taq.

I don't know anything about the Licor system.

 

I'd strongly suggest that you start by detecting serial dilutions of your probe spotted on a membrane, then switch to serial dilutions of your target spotted on a membrane, and only when that is working well (low background, high sensitivity) should you worry about running gels and blotting.






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