Washing In A Sandwich Box - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

Washing in a Sandwich
Box (continued)
Thermo Scientific
5. Remove the first wash solution and replace it with an equal volume of
the prewarmed second wash solution. Replace the lid and shake at the
required temperature for the required time period.
6. Step 5 should be repeated for any subsequent wash. The washing
protocol using the shaking platform will be identical to that used in
the bottles.
Note Refer to Appendix I for additional information on Washing.
In addition to bulk processing of blots during stringency washing steps,
the shaking platform is ideal for performing several other stages of the blot
generation and processing procedures. Three examples are given as follows:
1. Pre-treatment of Gels prior to Nucleic Acid Transfer
Gentle agitation of the gel during pre-treatment steps is essential to
prevent damage to the gel. Pre-treatment steps are usually performed at
ambient temperature.
(i) Depurination - 0.25 M HCI 10 minutes
(ii) Denaturation - 1.5 M NaCI, 0.5 M NaOH, 30 minutes
(iii)Neutralisation - 1.5 M NaCI, 0.5 M Tris CI pH 7.2, 30 minutes
2. Pre-washing of Filters prior to Colony Blot Hybridisation
Pre-washing is often advantageous to remove colony debris and thus
reduce background noise when screening bacterial colonies by
hybridisation. After lysing the bacterial colonies and fixing the DNA
on the membrane, pre-wash in a solution of 2 x SSC/0.1% SDS at
65°C. Use several changes of buffer.
After several washes, residual colony debris may be removed by gently
rubbing filter with a gloved finger.
3. Probe Stripping from Filters after Hybridisation
An example of probe stripping procedure is as follows: -
a) Wash the filter at 65°C in 5mM Tris CI pH8.0, 0.2mM EDTA,
0.1 x Denhardt's reagent for 1-2 hours.
b) Check for residual activity by autoradiography, then re-probe.
Section 4
Methodology of Hybridisation
Shake 'n Stack 4-5