Visualise integrity of RNA by UV illiumination/EtBr staining
1. Wearing gloves, remove gel from the tank, carefully place gel on transilluminator and take a photograph. It is often useful to place a fluorescent ruler on the gel prior to the photograph.
This aids later in matching the position of the ribosomal markers on the photograph (which may not be actual size) with the final X-ray film of the hybridized mRNA.
2. Use extra blue roll when transporting gel from tank to UV transilluminator. Take care not to spill the running buffer because it may contain ethidium bromide (EtBr).
Transfer to nylon membrane
1. Set up transfer as shown and leave to transfer by capillary action overnight.
2nd Day(AM) Synthesize and purify probe
The Northern blot can be probed with any labelled RNA or DNA. Here we will probe the blot with DNA which is radiolabelled. See appendix for details of other probes.
Materials required
2 x SSC buffer 50 µCi[a32P]dCTP, 3000Ci/mM
High Prime solution Screw cap vials
| Radioactivity-Caution β-particles are emitted by 32P radionucleotides need to be handled with care and disposed of in a designated radioactive waste bin Ð wear two pairs of gloves |
Transfer disassembly
1. Carefully disassemble the transfer apparatus and remove the nylon membrane.
2. Fix the RNA to nylon membranes using UV crosslinker.
3. Label your hybridization chamber
4. Place blot in hybridization chamber (RNA ’side’ facing upwards).
Probe synthesis
High Prime is a reaction mixture that contains random oligonucleotides, Klenow polymerase, labelling grade dATP, dGTP, dTTP, and an optimized reaction buffer concentration in 50% glycerol for rapid and efficient labeling of DNA with 32P- or 35S-labeled dCTP. The plasmid we will use contains the P2X2 cDNA. We use this plasmid as the template DNA with High Prime and [a32P]dCTP.
1. Place 25 ng template DNA (final volume 11 µl) in a screw cap vial.
2. Denature DNA by heating in boiling water bath for 10 min.
3. Chill quickly on ice.
4. Pulse spin tube in microcentrifuge.
5. Mix on ice: 25 ng denatured DNA
4 µl High Prime solution
5 µl 50 µCi[a32P]dCTP, 3000 Ci/mM
6. Incubate for 10 min at 37℃.
7. Stop the reaction by adding 2 µl 0.2 M EDTA and heat to 65℃for 10 min.
