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A small repository of synaptic protein information
Caution! In converting these protocols to HTML, some units have been scrambled.
Specifically, microliters (ul) often appear as milliliters (ml).
Caution! In converting these protocols to HTML, some units have been scrambled.
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.
acc 1/90
