|
imported>Xenbase |
| Line 1: |
Line 1: |
| Protocol submitted by VGP from Stukenberg lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1311&tabId=0]
| | ==Reporting a bug in Xenbase== |
| | If you find a bug in Xenbase, we would really like to know! |
| | However, it is very important that we can find your bug so that we can fix it. |
| | To help us find your bug, please fill out [http://xenbase.org/xenwiki/images/6/6e/Bug_report.txt this] form, and e-mail it to us! |
|
| |
|
| | | ==Bug Handling Process== |
| link to protocol page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm]
| |
| | |
| | |
| | |
| 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.
| |