1. Mix the following:
190
ml DMS buffer (50 mM sodium cacodylate pH 7, 1mM EDTA pH 8)
4 ml calf DNA (1 mg/ml)
50,000
cpm labeled DNA
2. Chill to 0¡C.
3. Add 5 ml of 10% DMS (1:10 dilution of 99% dimethyl sulfate with ethanol). Immediately close cap and mix by vortexing for
10-15 seconds. Incubate tube 5 minutes at 20¡C.
4. Add 50 ml DMS stop solution (1.5M sodium acetate, 1 M BME, 250 mg/ml yeast tRNA) chilled to 0¡C. Mix the solution by vortexing, and then add 750 ml of ethanol chilled to -20¡C.
5. Mix the solution by
vortexing, and then store the tube for 5 minutes at -70¡C in a dry-ice/ethanol bath.
6. Recover the DNA by
centrifugation at 12,000 g for 5 minutes at 4¡C in a microfuge.
7. Transfer supernÕt to DMS waste
bottle.
8. To the pellet of DNA, add
300 ml of 0.3 M sodium acetate chilled to 0¡C.
9. Close the top of the
tube, and vortex the solution. Add 900 ml of ethanol chilled to -20¡C. Close the tube, mix the solution by vortexing, and then store the tube
for 5 min at -70¡C.
10. Recover the DNA by
centrifugation at 12,000 g for 5 min at 4¡C in a microfuge.
11. Remove the supernÕt, and
add 1 ml of ethanol chilled to -20¡C. Close the tube, and mix the solution by vortexing.
12. Recover the DNA by
centrifugation at 12,000 g for 2 min at 4¡C in a microfuge.
13. Remove as much of the
supernÕt as possible, and then dry pellet of DNA in a lyophilizer for 5-10
minutes.
14. Resuspend DNA in
100 ml of 1 M piperidine.
15. Close tube top securely.
Centrifuge to deposit fluid on bottom.
16. Incubate tubes for 30
minutes at 90¡C.
17. Cool tube to RT.
Evaporate to dryness in a lyophilizer (1 hour). During this hour, prepare
sequencing gel.
18. Remove tubes and add 20 ml H20. Close caps and vortex 30 seconds to redissolve
DNA. Centrifuge briefly to deposit all fluid on bottom. Check all of labeled
DNA has been washed from walls of tubes by water and is redissolved in the
fluid.
19. Open tubes and evaporate
sample to dryness in lyophilizer (15 min).
20. Remove tube and add 20 ml H20. Close caps and vortex 30 seconds to redissolve
DNA. Centrifuge briefly to deposit all fluid on bottom. Check all of labeled
DNA has been washed from walls of tubes by water and is redissolved in the
fluid.
21. Open tubes and evaporate
sample to dryness in lyophilizer (15 min) If samples have oily look or took
abnormally long to lyophilize, redissolve DNA in 10 ml fo H20 and carry out an additional cycle of
lyophilization.
22. Add 10 ml of sequencing gel-loading buffer (98% deionized formamide, 10mM EDTA,
0.025% xylene cyanol, 0.025% bromophenol blue). Vortex to redissolve DNA. Centrifuge briefly to deposit all fluid on
bottom.
23. Close tubes and heat 1
min at 90¡C to denature DNA before
loading.