DNA and RNA Blotting and
This procedure is for blotting and hybridising a previously
untested set of samples and/or probe. Once the hybridisation and washing
conditions have been determined subsequent blots can be processed much
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.