RNA/Zeta Probe Dot Blotting protocol

RNA/Zeta Probe Dot Blotting protocol

1)Make up RNA (up to 20μg)dissolved in sterile H₂ O,TE or 0.5% SDS to 500μl with ice-cold sterile 10mM NaOH,1mM EDTA and apply it to Zeta Probe membrane,held in a dot-blot apparatus,which has been pre-wetted in 2 x SSC.

2)Apply a vacuum across the apparatus to draw the solution through the membrane and wash each dot once with 500μl ice-cold 10mM NaOH,1mM EDTA.

3)Remove membrane from apparatus and wash for 5 mins in 2 x SSC at room temperature.

4)Allow membrane to air dry.

At this stage the membrane can be stored dry at -20℃ until required for hybridisation to the probe of choice or it can be used immediately.