(Goodman Lab)
Stock Solutions
3M NaOAc, pH 5.2 with HAc (MW=82)
6M Guanidine Hydrochloride in 0.1M NaOAc, pH 5.2 (MWGuHCl=95.54)
5.7M Cesium Chloride in 0.1M NaOAc, pH 5.2 (MWCsCl=168.37)
Procedure
1. Brush embryos onto teflon pan with ddH2O and filter adults on nitex.
2. Rinse well with ddH2O to remove yeast.
3. Scoop into beaker.
4. Dechorionate with chlorox:ddH2O (60:40); swirl for 2-3'.
5. Pour onto nitex and wash thoroughly with ddH2O.
6. Allow embryos to settle 5-10' and aspirate off chorions.
7. Reapeat step 6 with clean ddH2O.
8. Pour dechorionated embryos onto nitex, wash with more ddH2O and dry briefly.
9. Weigh embryos (use max. 3g/prep).
10. Dounce by hand using the A pestle (1g embryos/10 ml GuHCl-NaOAc).
11. Spin 10' @ 10krpm in 30ml Corex tubes to remove cellular debris.
12. Harvest the supernatant and layer onto a 4ml CsCl cushion in polyallomer tubes (13ml) for the SW41 rotor. Cfg. 25krpm, 18h @ 18oC.
13. The RNA should appear as a gelatinous pellet at the bottom of the tube. Remove the upper 5-8ml CsCl-cell homogenate by aspiration or pipetting. Pour off the remaining supernatant and keep the tube inverted to avoid backwashing proteases on the RNA! Use a razor blade to cut away the upper half of the tube.
14. Carefully resuspend RNA in DEP-H2O (or TE+0.2% SDS) and NaOAc/EtOH ppt.
N.B. TLA 100.2 preps: layer 1.8ml GuHCl extract on a 0.3ml CsCl cushion; centrifuge 4h, 57 krpm @ 20oC.
