Preparation of TLC plate:
Cut a piece of PEI-TLC. Width = number of samples (including four markers) times 0.8 cm plus 1.2 cm.
Draw a line 2.3 cm from bottom with a soft pencil. Starting 1 cm from the edge, draw a cross for every 0.8 cm (place of loading).
Pre-develop in water. Let water develop all the way to the top.
Dry the plate completely. Cut off the top 2-5 cm (miscolored region).
Pre-develop in 0.225 M (NH4)2SO4 to 1-2 cm above loading line.
Dry plate completely.

Decapping assays:
10 µl:
≥10,000 cpm cap-labelled substrate
1 µl 10xCE buffer
Decapping enzyme or cell extract.
0.2 µl RNAsin
30C, 2h.
2 µl 0.2M EDTA
Load 1-5 µl of samples slowly onto dry pre-developed PEI plate so liquid does not float on top of plate. Load 20-30 µg of markers (GMP, GDP, m7GMP, m7GDP).
Develop in 0.225 M (NH4)2SO4 to 1-2 cm below top.
Dry plate completely.
Circle markers under UV light.
Wrap in Saram Wrap and expose to film (remember autorad tape for orientation).

Buffers:
10xCE buffer:
200 mM Tris pH 8.0
1 M KCl
10 mM MgCl2
10 mM DTT
0.5 mg/mL BSA

EC 050413