Thermo Scientific Shake 'n Stack 6244, Руководство по эксплуатации и техническому обслуживанию

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A-2 Shake ‘n Stack Thermo Scientific
Section 11
Appendix
Washing Procedure
Stringency washing should be carried out 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 wash progresses 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 106cpm/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. 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.
Background
Reduction (continued)