northern bloting help - radioactivity (May/07/2006 )

Hello to all of you,

I am going to see the expression of COX-2 mRNA at transcription level. And the strategy which I am going to follow is Northern blotting. And I don’t have much experience of this.

I am going to label the 20 nt long oligonucleotide with gamma p32 ATP( end labelling).

I have some doubt and queries, wishing to get answer on this bioforum,

1. I baked the northern blot at 80degree Celsius and now where I shd keep this, at -20 or -80 degree temp.

2. I am not going to use the end labeling kit, I will use do it manually with T4 polynucleotidase kinase. I am afraiding that will I get result or not with this. Any one who had done this manually can please guide me.

3.I have no experience with the use of radioactive material and their calculation of specific activity. Will somebody help me in this regard.


And some one have other nice suggestion for me , plz most welcome , I will be very much thankful for this.

hi
question 1 : you're referring as fixing nucleic acids on the membrane. 2h at 80° seems ok, but if you can, use also a UV crosslinker (or UV length at 254nm). Do UV cross link before baking membrane (i tell you that point for further exp). After that, i kee p the membrane dry in saran at -20°. For "thawing" 10' RT is sufficent.

question 2 : i currently do a northern blot with my mRNA. As i don't have the whole cDNA, i do an oligo T4 labellling, and uses the church buffer method. Briefly, i use for the mix : 10pmol of probe, 2.5µl gamma P32 aTP, 2µl of T4 PNK, in total reaction volume of 50µl. I purify the probe by a nucleotide removal kit (quiagen) or equivalent. Then 2h prehybridization and overnight hybridization in rollers at 37°. 3x5' of washes ad exposure.
Hybridization buffer : 0.5 M phosphate buffer pH 7.2, 10mM EDTA, 7% SDS
Washes buffer : 40mM phosphate buffer, 1% SDS

question 3 : after purifying the probe, i elute in 200µl and pick 1µl as an aliquot. If you can't purify your probe, dilute it up to 200µl and pick 1µl. Then with the aliquot, i do a 12% polyacrylamide 7M urea gel in TBE buffer and run my probe on it. The i allow the gel to be counted on an instant imager.
But when i didn't have that apparatus, i used a manual counter. As i know that 10pmol were labelled, and as i took 1/200 (means 50fmol) i can count my labelling. I'm not in the lab now, but i'll post the calculs i did.