Sara's CSF EXTRACT
¶ 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.
100mL CSF-XB + PIs:
¶ 2M Sucrose
¶ 40X Energy Mix
¶ Sperm Dilution Buffer (5X)
¶ 10X Calcium: 4mM CaCl2 in 1X Sperm Dilution Buffer
If the extracts will be spun onto coverslips, then also need:
¶ -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.
¶ Add1/1000 volume cytochalasin.
¶ 1/40 volume 40X energy mix.
¶ Add 1/40 volume 2M sucrose.
This page last modified 08/11/2011