Preparation of Xenopus egg extracts by dfsdf224s

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									                     Preparation of Xenopus Egg Extracts

From:
Blow J.J. (1993). J. Cell Biol. 122, 993-1002.
Chong, J.P.J. et al. (1997). Methods Enzymol. 283, 549-564.
Peter Gillespie (2002).

1. Egg production
Good quality eggs are essential for good extracts; this in turn requires fit and healthy frogs. The
   following is the current protocol
a) Frogs primed with 50 units Folligon (FSH; Intervet) 2 - 3 days before eggs required. This
   increases the number of stage 6 (mature) oocytes.
b) At about 4 p.m. on the day before eggs are required, frogs are injected with 400 - 600 units
   Chorulon (serum gonadotropin; Intervet), and are put in laying tanks containing High Salt
   Barth (110mM NaCl, 15mM Tris, 2mM KCl, 1mM MgCl2, pH adjusted to 7.6 with HCl) or
   MMR (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0.1 mM EDTA, 5 mM Hepes
   pH adjusted to 7.6 with NaOH) which inhibits premature activation of eggs once laid. The
   tanks are kept at <=23°.
c) Eggs from different frogs are collected in 600ml beakers. Any that are activated or are sick
   are removed with a pasteur pipette.

2. Activated egg extracts (LSS).
a) Eggs are dejellied in 2.2% cysteine monohydrate HCl, pH 7.8, 1mM EGTA. Dejellying is
   complete when the eggs pack together without any transparent gaps.
b) Eggs are rinsed three times in Barth solution (88mM NaCl; 2mM KCl; 1mM MgCl2; 15mM
   Tris HCl, pH 7.4, 0.5mM CaCl2) at room temperature.
c) Eggs in about 100ml Barth solution are activated briefly by adding 10 µl A23187 (20 mg/ml
   stock in DMSO). The eggs should roll animal-side up and the pigment should start to
   contract. After 5 mins the A23187 is washed away with three more rinses in Barth solution.
d) Eggs are then washed three to five times in Extraction Buffer (50mM KCl; 50mM Hepes
   KOH, pH7.6; 5mM MgCl2; 2mM DTT) at room temperature.
e) Eggs are transferred to a 15ml Falcon tube, and are packed by centrifuging at 3000 rpm in
   an Optima refrigerated centrifuge, using an JS13.1 (swinging bucket) rotor (approx 800g) for
   1 min. As much buffer as possible is then removed from the packed eggs. Sick eggs float to
   the surface after this treatment, making the surface appear white; these should be removed
   with a pasteur pipette.
f) Eggs are then spin-crushed by centrifuging at 12000rpm for 10 mins in the same rotor
   (approx 12000g). This separates the eggs into a grey insoluble pellet, a brownish cytoplasm
   and a yellow lipid plug. The cytoplasm is taken from the top with a pasteur pipette or by using
   a 20-G needle and 1-ml syringe via side puncture; some contaminating lipid is tolerated at
   this stage. From this point on, keep the extract at 4°C or on ice.
g) Extract is transferred to polyallomer SW55 tubes (Beckman); for the sake of good separation,
   the tubes are not more than half-filled. Extract is then supplemented with 10µg/ml
   cytochalasin B (from 10mg/ml stock in ethanol) and 15% (by volume) Extract Dilution Buffer
   (EDBS: 50mM KCl, 50mM HepesKOH pH 7.6, 2mM DTT, 0.4mM MgCl2, 0.4mM EGTA,
   1µg/ml each of pepstatin, leupeptin, and aprotinin, 10% sucrose). This aids further
   separation.
h) Extract is cleared by centrifuging at 20,000rpm in an SW55 rotor at 4° for 20 mins. Harder
   spins give extracts more stable at 4°; softer spins give extracts better at replicating naked
   DNA. The spin usually yields a small black insoluble pellet, a small quantity of clear
   gelatinous material (glycogen+ribosomes), a larger loose brown membranous pellet above it
    (mitochondria), a clear golden cytoplasmic fraction, a variable amount of white membranous
    material floating in this, and a small yellow lipid plug. This time only the clear golden layer,
    including the floating white membrane layer, is taken. In particular, the loose membranous
    pellet (containing mitochondria) should be avoided as it decreases extract longevity on
    freezing. If this is not possible, the extract can be respun under the same conditions. NB
    extracts activated for short times (<5mins) are harder to clear.
i) The extract is then passed through a 25µm nylon mesh (eg Nybolt), mounted in a screw-cap
    Eppendorf with a large hole cut through it. Extract should flow under gravity, but can be
    forced through with the plunger if necessary. This gets rid of any particulate junk.
j) The extract is then mixed with 1% v:v glycerol. 4 plastic petri dishes are then filled with liquid
    nitrogen, and using a siliconised yellow tip with the end cut off, 20ml aliquots are dropped
    into each dish in turn.
k) The beads are collected with Millipore forceps and are stored under liquid nitrogen. They are
    stable for >5 years.

3. Unactivated Egg Extract (ULSS, CSF-arrested extract).
To make unactivated extract, egg quality is crucial, since even a small proportion of activated
    eggs will release the CSF block present in all the others. Activated eggs must be removed
    before the packing spin; they can be identified because they have lost the clear
    circumferential distinction between animal and vegetal poles.
a) Eggs are dejellied in 2.2 % cysteine, pH 7.6, 1mM EGTA. They are then rinsed in High Salt
    Barth or MMR, and then washed in Unactivating Extraction Buffer (UEB: 50mM KCl; 50mM
    Hepes KOH, pH7.6; 5mM MgCl2; 5mM EGTA; 2mM DTT).
b) Eggs are transferred to a 15ml Falcon tube, containing 1 ml of UEB plus protease inhibitors
    (10 µg/ml each leupeptin, pepstatin and aprotinin) and 100 µg/ml cytochalasin D. Eggs are
    packed by centrifuging at 3000 rpm in an Optima centrifuge, using an JS13.1 (swinging
    bucket) rotor (approx 800g) for 1min at room temperature. As much buffer as possible is then
    removed from the packed eggs. Sick eggs float to the surface after this treatment, making
    the surface appear white; these should be removed with a Pasteur pipette.
c) Eggs are then spin-crushed by centrifuging at 12000 rpm for 10 mins in the same rotor
    (approx 12000g) at room temperature. This separates the eggs into a grey insoluble pellet, a
    brownish cytoplasm and a yellow lipid plug. Collect the cytoplasmic layer carefully using a
    20-G needle and 1-ml syringe via side puncture. From this point on, keep the extract on ice.
e) Supplement with: 10 µg/ml protease inhibitors (10 µg/ml each leupeptin, pepstatin and
    aprotinin), 100 µg/ml cytochalasin D and 15% LFB1/50 (40 mM Hepes KOH, pH 8.0; 20 mM
    K2HPO4/KH2PO4, pH 8.0; 2 mM MgCl2; 1mM EGTA; 2 mM DTT; 10% sucrose; 1µg/ml each
    of leupeptin, pepstatin, and aprotinin; plus 50 mM KCl), and mix gently.
f) Transfer to SW50 tubes, with a maximum of 3 ml per tube. Centrifuge at 30000 rpm in a pre-
    cooled SW55 rotor for 20 mins at 4º.
g) Remove the lipid plug from the top of the tube and collect all of the golden cytoplasmic layer
    (including the ‘wispy’ membrane layer below the lipid plug). Do not disturb the ‘dirty yellow’
    membrane layer below the cytoplasm; this layer contains the mitochondria which will lyse
    after freeze-thawing the extract and will promote apoptosis.
h) The extract is then passed through a 25µm nylon mesh (eg Nybolt), mounted in a screw-cap
    Eppendorf with a large hole cut through it. Extract should flow under gravity, but can be
    forced through with the plunger if necessary. This gets rid of any particulate junk.
i) Add 1% v:v glycerol and mix gently. 4 plastic petri dishes are then filled with liquid nitrogen,
    and using a siliconised yellow tip with the end cut off, 20ml aliquots are dropped into each
    dish in turn.
j) The beads are collected with Millipore forceps and are stored under liquid nitrogen. They are
    stable for >5 years.

								
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