30µl Reaction:
2µl Capping Buffer
4µl α32PGTP (3000Ci/mmol)
2ug mRNA
0.5 µl RNAse Out
4µl capping enzyme (fraction 6)
1.65 µl SAM (.66mM)
~15 µl water

Incubate at 37 C for 2 h.

Run through G-50 column
Add 5 uL of EDTA

Run on 8% poly acrylamide/6M Urea Gel

40ml per gel
0.5X TBE
6M Urea (14.4g)
8% polyacrylamide (19:1 acryl:bis) 8ml
Mix gently (air will strongly inhibit polymerization)
40 µl TEMED
400 µl 10% APS

Pre run at 20-25 W for at least 30 min.

Add 25 µl Formamide Load Buffer to sample
Heat at 95 oC for 1 Min.
Load on Gel
Run gel at 19-20W

Place film on gel (on saran wrap) for ~1 min. use biorad tape to orient.
Cut out capped RNA
Add 400 µl .3M NaAc pH 6, 5mM EDTA, 0.05% SDS
400 µl PCA
Nutate over night

EtOH precipitate and dissolve in 20-40 µl ddH20
Count 1µl in scintillation counter

Capping buffer (make 15X)
750 mM Tris pH 7.5
90 mM KCL
37.5 mM DTT
18.8 mM MgCl2
1.5 mg/ml BSA

EC 050413