Dot blot for determininig biotinylation of 4-thiouridine RNA

Hybond-N, nylon membrane

Streptavidin-HRP (R&D systems Cat :890803)

PBS x10 

20% SDS

0.5M EDTA

ECL kit

X-ray Film

Stratalinker UV cross linker

benchtop shaker

film developer

1. Cut the nylon membrane to the desired size. 

2. Place in plastic box/plate suitable for washes

3. Write, with a pencil - not a pen - the date and label the intended spots

4. Take 2-4 ul of RNA in water and place on membrane. Make sure there is enough distance between samples. Default amount of RNA is 1 ug/spot, but can go lower or higher according to needs.

5. Take membrane to "stratalinker" UV cross linker. Cross link, twice, with 1200 J. Remove any plastic lid prior to crosslinking.

6. Wash membrane in blocking solution, room temp (RT), 20 min with mild shaking

7. Incubate with streptavidin-HRP (1:500 in blocking solution), RT, 15 min with shaking.

8. Wash twice with Wash buffer 1, RT, 10 min with shaking

9. Wash twice with Wash buffer 2, RT, 10 min with shaking

10. Wash twice with Wash buffer 3, RT, 10 min with shaking

11. Use ECL to for detection according to standard protocol.
    

Blocking buffer: PBS x1 pH 7.4, 10% SDS, 1mM EDTA

Wash buffer 1: PBS x1 pH 7.4, 10% SDS

Wash buffer 2: PBS x1 pH 7.4, 1% SDS

Wash buffer 3: PBS x1 pH 7.4, 0.1% SDS