Preparation of cycling egg extract (Shechter lab): Difference between revisions
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Sterile-filter | Sterile-filter | ||
Other ingredients - Ca2+ ionophore A23187 | Other ingredients - [[calcium ionophore|Ca2+ ionophore A23187]] | ||
5mg/ml aprotinin/lepeptin | 5mg/ml aprotinin/lepeptin | ||
1000 Units/ml HCG hormone | 1000 Units/ml HCG hormone | ||
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7. Activate with Ca2+ ionophore A23187 at 0.2 ug/ml (5ul stock /10ml volume)Set timer immediately: Leave ~4-5minutes, until cortical rotation/activation observed (clearly obvious rotation of animal pole visible by appearance of a dark field of rotated eggs) | 7. Activate with [[calcium ionophore|Ca2+ ionophore A23187]] at 0.2 ug/ml (5ul stock /10ml volume)Set timer immediately: Leave ~4-5minutes, until cortical rotation/activation observed (clearly obvious rotation of animal pole visible by appearance of a dark field of rotated eggs) | ||
8. Rinse with 1X XB four times and leave eggs at room temperature, remove bad eggs | 8. Rinse with 1X XB four times and leave eggs at room temperature, remove bad eggs | ||
Latest revision as of 07:43, 28 May 2015
Protocol submitted by VGP from David Shechter lab protocols [1]
Link to PDF: [2]
Recipes:
XB - 50mM Sucrose 2mM MgCl2 100mM KCl 10mM Hepes-KOH pH 7.8 0.1mM CaCl2 Sterile-filter
MMR - 100mM NaCl 2mM KCl 1mM MgCl2 2mM CaCl2 0.1mM EDTA 10mM Hepes-KOH pH 7.8 Sterile-filter
Other ingredients - Ca2+ ionophore A23187 5mg/ml aprotinin/lepeptin 1000 Units/ml HCG hormone 100 Units/ml PMSG hormone 2% L-cysteine, pH 7.7 (adjusted with KOH)
Procedure:
Notes: cycling extract is exceedingly time sensitive, use immediately when prepared. Observe cycling of nuclei with DAPI staining under microscope
1. Induce egg laying as usual with 800 Units HCG, late in the day (~15 hours prior to extract prep); keep frogs in 100mM NaCl or 1X MRR.
2. Squeeze eggs into 1X MMR in a petri dish in a cool laboratory room (<22 ˚C)
3. Rinse eggs with 1X MMR, remove bad and necrotic eggs; if there are too many “bad” eggs, discard the entire batch
4. Rinse eggs with ddH2O in order to promote Ca2+ vesicle swelling - leave for 10 min
5. Dejelly eggs with 2% cysteine in 1X XB Buffer, pH 7.8 for 3-4 minutes, watch for refractive jelly coats floating off into buffer
6. Rinse with 0.2X MMR two times, aspirate remaining buffer carefully with a transfer pipet
7. Activate with Ca2+ ionophore A23187 at 0.2 ug/ml (5ul stock /10ml volume)Set timer immediately: Leave ~4-5minutes, until cortical rotation/activation observed (clearly obvious rotation of animal pole visible by appearance of a dark field of rotated eggs)
8. Rinse with 1X XB four times and leave eggs at room temperature, remove bad eggs
9. Add cytochalasin B to 2.5 ug/ml
10. Pack eggs at 800 rpm x 30 sec, remove excess buffer carefully
11. Incubate at room temperature (<22 ˚C) until timer reaches 30 minutes (timer started in step 7)
12. Transfer to ice for 15 minutes
13. Crush at 10,000RPM x 10 minutes at 4 ˚C
14. Remove extract (either by side puncture with a wide bore needle or by using a metal spatula to push away yolk and collecting middle layer with a blue-tip pipet) and respin 10,000RPM x 10 min. Remove clear extract with a pipet.
15. Proceed with experiment immediately, consider as time=0.