Northern Blotting - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

Northern Blotting

Electrophoresis of RNA should be carried out in a denaturing gel system. Maniatis et al
1989 gives details of running denaturing RNA gels using formaldehyde or glyoxal.
1. After electrophoresis of the RNA in a denaturing gel the transfer can be set up as
described for DNA gels (see Southern Blotting above). The denaturating step 3 and
the neutralising step 4 of the gel are not required, as the RNA is denatured in the
gel. However, some researchers recommend reduced stringency denaturation and
neutralisation steps to facilitate transfer of large RNA molecules as follows: -
Gentle agitation of the gel is essential to prevent damage to the gel during these
steps. The shaker in the Shake 'n' Stack, Midi Dual 14 and Maxi 14 are ideal for
this purpose.
2. RNA transfer is carried out in 10-20 x SSPE, using the same procedure as outlined
for DNA gels in Southern Blotting section above.
3. After transfer of the RNA to the Hybridisation membrane is completed, fixing of the
RNA is carried out by baking at 80°C for 2 hours, or by UV cross-linking.
4. After fixing the RNA, the membrane is ready for Hybridisation. Membranes not
used immediately may be stored between sheets of Whatman 3MM paper in
sealed plastic bags at 4°C.
© Thermo Scientific, May 2003. Issue 7
50mM NaOH, 10mM NaCl
100mM Tris HCl Ph7.5
30 minutes
30 minutes
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