Decapping Assay Protocol

1µl 200 µM cap analog
1µl cap-labeled RNA (50-100 k)
4µl dH2O
1µl 10x CE Buffer
4µl extract (hela)

Incubate for 30°C for desired time course

Stop reaction with 1µl 0.5 M EDTA

Bring up volume to 20µl with dH2O

Phenol extraction

Remove 10µl of aqueous phase

Add 4µl RNA buffer

Incubate at 90°C for 30 seconds

Load on 20% gel (19:1 acryl to bis-acryl)

Allow bromophenol blue to migrate about half way down the gel

 

20% Polyacrylamide Gel

12g urea
20ml 40% acrylamide
4ml 10x TBE
400µl 10% APS
30µl TEMED

 

10x CE Buffer

500mM Tris pH 7.9
300mM (NH4) 2 SO4
10mM MgCl2