Since I get an extreme amount of products when doing PCR and 3'-UTR RACE for my gene of interest I am thinking of turning to the old-fashioned northern blot to increase sensitivity. I need to validate if my PCR and RACE results (which seem to be bona fide, but it would be nice to validate them in a different way). However, I was born in 1985 and being a relatively young molecular biologist, I never consciously encountered anybody that knows how to do northern blotting. So, if somebody has an extended protocol that explains all the steps, includes troubleshooting and tells me the the do's and the dont's in such a way that a newbie can follow it, my gratidude would be eternal.
We did that a large number of years ago following the procedure from Maniatis Sambrook & Russel. I recall being very happy with that protocol. The book is a good reference for just about anything that doesn't come in "kit" form.
Not sure if it's on the internet. You may have to find a library and pick up enormous pieces of paper (some archaic data storage method invented shortly after the stone tablet!)
Yes, the papyrus tomb is titled "Molecular Cloning", and the Northern blot protocol works nicely (note: I know it from the 80's). A new edition came out in 2012, Molecular Cloning, edited by Green and Sambrook, and it still includes Northern Blotting.
Of course, Maniatis! I hope it includes a version that doesn't need radioactive probes.
When RT-PCR was introduced, some people (including myself) did RT-PCR Southern blotting (Southern blot of RT-PCR product) It will be far easier than northern blot..Consider doing that if it works for your need...
Hi,
I still see northern blotting results being published in high impact factor papers, hence i don't take it as ''old fashioned'' yet. I included my own protocol for you in the attachments, I hope you find it helpful. BTW we use THERMO kit for northern blot in our lab.
Thanks Shirin! I am sorry to have used the term old-fashioned. Maybe established would be a better way to describe this technique :)
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