DNA and RNA Blotting and Hybridisation

• DNA samples are run on gels according to any standard protocol. This protocol for RNA blotting refers to glyoxal RNA gels.

 
• DNA gels are soaked for 15min in 0.4M NaOH, 1.5M NaCl and then for a further 15 min in 0.5M Tris/HCl, pH 7.6, 1.5M NaCl

 
• Glyoxalated RNA gels are used immediately with no other treatment.

 
• Assemble a blotting tank consisting of a polypropylene tray containing 400ml 10X SSC, covered with a glass plate over which two layers of Whatman 3mm paper, presoaked in 10X SSC, have been laid so as to allow the paper ends to wick into the SSC in the tank below.

 
• Slide the gel onto the centre of the 3MM paper and place parafilm around the gel to prevent wicking of SSC around the edges of the gel.

 
• Lay Nylon Membrane (Gellman, Biodyne B) cut to exactly the same size as the gel and prewetted in 2X SSC over the gel. Take care to expell all bubbles and do not move the membrane once applied.

 
• Lay over two sheets of dry 3MM paper cut to the same size and cover with a 5cm layer of green paper towels.

 
• Put a glass plate over the towels and place about  a 0.25kg weight (eg about half a 500ml bottle) onto the plate. Allow to transfer for 4 to 16 hours. Genomic blots and RNA blots are generally left for longer. Simple restriction digest-type Southerns can be transferred for shorter times. Blots for quantitative or semi-quantitative anlysis should contain suitable standards for calibration and inter-blot comparison.

 
• After transfer period, disassemble the apparatus and remove and carefully mark membrane for orientation. Rinse for 10 seconds in 2X SSC and bake for 1 hour at 80°C.

 
• Use Techne ovens and hybridisation tubes. Prehybridise in up to 50ml of 0.1M sodium phosphate, pH 7.7, 7% SDS at the required temperature (50- 65°C) for 1-3 hours. Remove prehybridisation solution and add minimal amount of 0.1M sodium phosphate, pH 7.7, 7% SDS (ideally 0.1ml /cm2 membrane).

 
• Add denatured, 32P lableed probe and hybridise at required temperature for 4 to 24 hours. Genomic and RNA blots should be hybridised for longer than simple Southerns.

 
• Carefully discard or collect hybridisation by pouring out of tube and add 50ml of 1X SSC. Wash for 15min at hybridisation temperature. Repeat twice. Check membrane radioactivity using a Geiger counter. If low (<50cpm), blot filter damp-dry on tissue paper and place on an old autorad film. Cover with plastic film and autoradiograph. If counts are high repeat wash using 0.5X SSC, then check. If still high reduce to 0.1X SSC before autoradiography.

 
• If blots are not allowed to dry completely they can be rewashed after developing the autoradiograph and then autoradiographed again.

This procedure requires the use of radioactive isotope and all prospective users should be registered isotope users and have attended a course in handling radioisotopes. Specific details can be obtained from departmental radiation protection officers (Douglas Tocher for the Institute of Aquaculture) and specific instruction should be sought from experienced personnel. Radioisotopes may only be used in designated areas and their ordering, use and disposal must be authorised and logged according to strict procedures.