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

Page: 79 / 92

© Thermo
Scientific
, May 2003. Issue 7 26
Washing Procedure
Stringency washing should be carried out as follows, using large volumes (approximately
100ml) of the following solutions which should be pre-warmed to the required
temperature: -
1. 2 x 15 minutes with 2 x SSPE (SSC), 0.1% SDS at 65
°
C
2. 1 x 30 minutes with 2 x SSPE (SSC), 0.1% SDS at 65
°
C
3. 1 x 10 minutes with 0.1 x SSPE (SSC), 0.1% SDS at 65
°
C
All wash solutions should be pre-warmed to the appropriate temperature. An initial room
temperature wash is not recommended and can cause background problems.
The final wash is a high stringency wash. Use of a hand held monitor to give an
indication of the decrease in radioactivity as the washes progress is recommended and
should give some indication as to whether this final wash should be carried out.
In general terms, the stringency of Hybridisation and washing steps is increased by
increasing the temperature, or by decreasing the salt concentration. Hybridisation should
be carried out under relatively low stringency conditions compared to the washing
procedures. It is generally simpler and more effective to adjust the stringency during the
washing steps by altering the salt concentration rather than the temperature.
Probe Preparation
The final probe concentration should be in the region of 25-50ng/ml of Hybridisation
solution, at approximately 1-5 x 10
6
cpm/ml.
The optimum length of probe is approximately 500-800bp. Purification of the labelled
probe to remove unreacted triphosphates will reduce background problems, and is
recommended for all Hybridisations - Thermo Recovery kits are excellent for this purpose.
Probe solutions should be pre-warmed to the Hybridisation temperature and care should
be taken to ensure the membrane is not exposed to the concentrated probe solutions if
adding it directly to the bottles.