Thermo Scientific Shake 'n Stack 6244, Operating And Maintenance Manual

Page: 64 / 92

© Thermo
Scientific
, May 2003. Issue 7 11
Northern Blot RNA Hybridisation
1. Prehybridisation is carried out by incubating the membrane in Northern Blot
Prehybridisation Buffer (recipe on page 33). Denature salmon sperm DNA by
boiling for 5 minutes and then chilling on ice. Add the denatured salmon sperm
DNA to the buffer, to a final concentration of 100
µ
g/ml.
2. Incubate with agitation or in a rotisserie for at least one hour at 42
°
C.
3. The volume of prehybridisation buffer required varies according to he Hybridisation
system being utilised. In general terms, the minimum volume of buffer should be
used such that the membrane is covered by the fluid at all times (approximately
0.1ml/cm
2
), or if in a Hybridisation bottle, 10-20ml for a large bottle and 5-10ml for
a small bottle.
4 . Denature the labelled probe by heating to 100
°
C and incubating for 5 minutes. Chill
on ice and add to the prehybridisation solution. Depending upon the system
utilised, the probe may be added directly or, alternatively, some prehybridisation
buffer is removed, the probe added to this, and then the solution replaced in the
Hybridisation vessel. Some researchers may prefer to use fresh Hybridisation
solution.
5. Hybridise with agitation, or rotating in bottles, for approximately 12 hours at 42
°
C.
6. Stringency washing steps are carried out as follows using large volumes (at least
50ml) of the following solutions which should be pre-warmed to the required
temperature: -
2 x 15
minutes with 2 x SSPE 0.1% SDS at
42
°
C
1 x 30
minutes with 1 x SSPE 0.1% SDS at
42
°
C
1 x 15
minutes with 0.1 x
SSPE 0.1% SDS at
42
°
C