CSF extract, sara's protocol (Stukenberg lab): Difference between revisions
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Revision as of 08:42, 15 November 2011
Protocol submitted by VGP from Stukenberg lab protocols [1]
link to protocol page [2]
Sara's CSF EXTRACT
Preparation:
2-3 L of 1X MMR, make night before, store at 20°C.
200mL de-jelly solution, 2% cysteine, pH to 7.8 with 0.9mL 10N NaOH.
750mL XB: 10mM K-HEPES 50mM sucrose 1X XB 11uL 10N KOH per 100mL to keep pH 7.7
250mL CSF-XB: 250mL XB (from above) 5mM EGTA 1mM MgCl2
100mL CSF-XB + PIs: 100mL CSF-XB (from above) 10 ug/mL LPC
2M Sucrose
LPC
Cytochalasin
40X Energy Mix
Sperm
Extract Fix: 60% Glycerol 1X MMR 1ug/mL Hoescht 10% Formaldehyde
Sperm Dilution Buffer (5X)
10X Calcium: 4mM CaCl2 in 1X Sperm Dilution Buffer
If the extracts will be spun onto coverslips, then also need:
Fixation Buffer: BRB80 20% Glycerol 0.5% TX-100 4% Formaldehyde
Cushion: BRB80 30% Glycerol
-20°C MeOH
Making a CSF Extract
Collect eggs.
Wash eggs in MMR 2-3X.
Remove as much MMR as possible, and pour on dejellying solution.
De-jelly 7-10 min. Rinse 1X in de-jelly. While eggs are de-jellying, add 1mL CSF-XB +PIs to centrifuge tubes. Add 10uL 10mg/mL cytochalasin and flick tube well.
Wash de-jellied eggs 3-4X in XB.
Wash eggs 2-3 X in CSF-XB.
Wash eggs 2X in CSF-XB + PIs. Leave in small volume of CSF-XB+PIs.
Gently pipet eggs into centrifuge tubes, put pipet below meniscus.
Remove excess buffer.
Spin 10 sec at 1500 rpm (setting 4) in clinical centrifuge/
Aspirate off buffer. Add 100uL Versilube to top
Pack eggs: spin at 2000rpm (setting 5) for 30 sec and full speed for 1min in clinical centrifuge.
Aspirate off versilube and buffer from top of eggs.
Centrifuge packed eggs at 10,000 rpm for 15 min at 16°C in the SW55.1 rotor.
Collect cytoplasmic layer of extract with 18-guage needle into a 1-cc syringe. Avoid bubbles.
Transfer to new tube and estimate volume.
Add 1/1000 volume LPC.
Add 1/1000 volume cytochalasin.
1/40 volume 40X energy mix.
Add 1/40 volume 2M sucrose.