UV cross-linking (9/22/98)
1.
Set up a 25 µL binding reaction:
20 to 35 µg NE in a total vol. of 15 µL buffer DG
1 µL nonspecific competitor (10 mg/mL tRNA or 0.3 to 10 mg/mL heparin)
1 µL 55 mM MgCl2
1 µL 10 mM ATP
1 µL 0.5 M creatine phosphate
5 µL nH2O
2.
Preincubate for 8 to 10 minutes at 30˚ C.
3.
Add 1 µL RNA probe (100,000 to 200,000 CPM) and continue the 30˚ C incubation
for 15 minutes.
4.
Quick chill binding reaction on an ice-cold aluminum block.
5.
Irradiate with a 254 nm UV illuminator at 2.2 mW/cm2 for 8 to 12 minutes
(approximately 4.5 mm between the UV lamp and the top of the liquid).
6.
RNase digestion for 30 minutes at 30˚ C with 1-2 µg RNase A (and 10 U RNase T1).
7.
Add 5.2 µL 6 X SDS sample buffer [350 mM Tris-Cl, pH 6.8; 30% (w/v) glycerol;
10.3% (w/v) SDS; 0.6 M DTT; 0.012% (w/v) bromophenol blue] and boil for 5
minutes.
8.
Load ≈ 1/2 of the reaction or 16 µL on a 10 or 12% SDS-polyacrylamide gel.
For RNA-protein cross-linking within a gel slice.
1.
Place gel slice on an ice-cold glass plate (approximately 3-4 mm from the UV
illuminator).
2.
Irradiate at 3.5 mW/cm2 for 10 to 16 minutes.
3.
Digest with ≈ 50 µL RNase A (10 mg/mL) for 30 minutes at 37 ˚C with shaking at
250 RPM.
4.
Add 10 µL 6 X SDS sample buffer and incubate as in step 3.
5.
Boil for 5 minutes and analyze by SDS-PAGE.