Electrophoresis and transfer:
Dry 20 ug of RNA with the speed vac and resuspend in 10ul ambion gel loading buffer II.
Gel preparation: 15 % TBE/Urea/polyacrylamide gel
|3.75ml||7.5ml||11.25ml||40% Acrylamide 19:1|
Heat the mix at 37 °C to dissolve.
Adjust vol. to 10 mL per gel
Add 80 uL APS 10 % and 8 uL temed per gel
Heat samples 5min @65°C
Run at 200 V in 0.5 X TBE until the first blue dye is at the bottom of the gel. (1h)
Stain with EtBr (1ul/25ml MQH2O, 2X H2O wash) for 15 min and take a picture for loading control.
Transfer on Hybond XL membrane with the BioRad semi-dry apparatus in 0.5 X TBE at 0.4 A for 50 min.
UV-crosslink membrane X2 and store wrapped at 4C until ready to probe.
Probe preparation (star fire kit):
1 uL starfire probe (0.5 uM)
1 uL template oligo
1 uL starfire reaction buffer 10X
Incubate 1 min at 95 °C
Remove the tube from heat and allow the reaction to cool to RT (5 min). Briefly spin the reaction.
Add 3 uL of α32P dATP and 1 uL of exo- Klenow DNA polymerase
Incubate 1 hour at RT.
Add 40 uL of stop buffer
Prep oligo columns – 1min @1000g, discard FT
Load entire labeling reaction (47ul) onto Sephadex G25 oligo spin columns (Roche) and centrifuge 4min at 1000g.
Before use heat probe at 65 °C 5 min and keep on ice.
Pre-hybridization is done at 65°C for 1 h with 6 mL of Ambion UltraHyb oligo buffer.
Add 50 uL of starfire probe to the pre-hybridization buffer and shift the temperature of the oven to RT. The blot is then allowed to hybridize O/N.
The actual temperature of the oven the next morning is about 32 °C.
Washing and exposure:
Washes are performed in the oven at RT
2X 30 min washes in 0.5 % SDS
1X 15 min wash in 1X SSC, 0.2 % SDS
Membranes are exposed O/N or longer depending on the signal!
Source: Wu et al. Mol Cell 2010