CSF Extract Protocol
- Five days prior to
planned extract date, prime frogs with PMSG, 50 units/frog (this
dramatically improves the amount of extract l).
- Approx 16hrs before
preparing extract, inject frogs with Human Chorianic Ganadotropin (HCG), 600
- Place 4-6 liters of H2O
in 20 °C
- If you will be
collecting laid eggs place the frogs in buckets containing MMR. If only
using fresh eggs leave the frogs in water overnight. Collecting only fresh
or squeezed eggs improves egg quality but severely compromises the amount of
eggs that will be collected.
- Come in to lab early,
enjoy some fresh coffee and begin to set-up for making extracts. Prepare:
2-3 liters of MMR: 5 mM Na-HEPES
(pH 7.8), 0.1 mM EDTA, 100 mM NaCl, 2 mM KCl, 1 mM MgCl2,
2 mM CaCl2.
Prepare from a 10x or 25x stock
750-1000ml XB: 10 mM K-Hepes (pH
7.7), 100mM KCl, 1 mM MgCl2,
0.1 mM CaCl2,
50 mM sucrose. Maintain pH of 7.7 w/ 11
of 10N KOH per 100 ml. Make with 20x XB salts, 2 M sucrose, 1 M K-Hepes (pH
250 ml CSF-XB: XB + 1 mM MgCl2
+ 5mM EGTA. Take 250 ml of XB that was just made and add MgCl2
100 ml CSF-XB + Protease
Inhibitors: CSF-XB + 10
LPC. Use 100 ml of CSF-XB just made.
200 ml de-jellying solution: 2 %
(w/v) cysteine in 1x XB salts, pH to 7.8 with 0.9 ml of 10N NaOH. Use 20x XB
salts and cysteine. DO NOT PREPARE CYSTEINE UNTI LIMMEDIETLY BEFORE IT IS
- Collect eggs in fresh
MMR; good CSF and cycling extracts depends on the quality of eggs
collected. Always take care to handle eggs and extract very gently.
- Remove white puffy
eggs, stringy eggs, eggs that are mis-shapen and those that generally don’t
look bad. It is generally better to compromise quality for quantity.
- Wash eggs 2-3 in MMR.
Pour off as much MMR as possible.
- Again, remove any
puffballs and/or poor quality eggs.
- Add de-jellying
solution to eggs, intermittently swirling gently. De-jellying takes 5-10
minutes. After they are dejellyed, there volume will decrease up to five
fold and they will pack much tighter.
- Pour off de-jellying
- Wash de-jellied eggs
2-4 times with XB.
- Wash eggs 2-3 times
- Wash eggs 2 times with
CSF-XB + PI's.
- Transfer eggs to a 2.2
ml thin, clear plastic centrifuge tube containing 2
of LPC and 10
of cytochalasin B. The size of the tube depends on the number of eggs
collect. For lower numbers of eggs, use a smaller tube.
- Aspirate excess buffer
off of the eggs.
- Place in a round
bottom culture tube and spin in a clinical centrifuge on low for 30 seconds.
- Aspirate off as much
buffer as possible and add 100 - 200
of versalube oil.
- In clinical centrifuge
spin for 30 seconds on low and 60 seconds on high.
- Aspirate ALL
buffer and versalube from the top of packed eggs.
- Transfer the tube to a
HB-4 rotor and spin at 10,000 rpm, 15 minutes, 16
(or the equivalent in a similar spinning bucket rotor)
- Store the crushed eggs
on ice. The top yellow layer is the lipid layer and the dark bottom layer
is yolk and nuclei. The middle layer is the desired cytoplasmic extract.
- After wiping the
outside of the tube with a alcohol wetted kimwipe, insert an 18-gauge needle
into the side of the tube (BE CAREFUL), and draw out the cytoplasmic layer.
Use a new needle for each tube.
- Place the fresh
extract into a clean pre-chilled tube on ice.
- Estimate the volume of
extract obtained and add 10mg/ml
LPC, cytochalasin, 1x final concentration energy mix, and 1/40 volume of 2 M
sucrose. Add into cap and then tip a couple of times to mix.
- Test to determine if
the extract is arrested in mitosis. To do so, take 15
of fresh extract and to a tube that contains 1
of diluted sperm for experiment. Remove 7.5
and add to a tube containing 1
of 4 mM CaCl2.
Place at room temperature for 20-30 minutes.
- Place 1 ul of extract
onto a plane glass slide and overlay with 3
of extract fix (100
10x MMR, 0.1 ml
Hoechst, 300 ml
37% formaldehyde, 600
of 80% Glycerol). Examine under the microscope. The sample containing
calcium should exit mitosis and have interphase looking nuclei (rounded
morphology). The sample without calcium should remain in mitosis and contain
condensed mitotic chromatin.