Preparation of cycling egg extract (Shechter lab) - Revision history

imported>Kevin at 14:43, 28 May 2015

2015-05-28T14:43:48Z

← Older revision		Revision as of 14:43, 28 May 2015
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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

imported>Kevin at 14:42, 28 May 2015

2015-05-28T14:42:44Z

← Older revision		Revision as of 14:42, 28 May 2015
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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

imported>Virgilio: moved Shechter Lab - preparation of cycling egg extract (extract that proceeds through multiple rounds of mitosis/interphase) to preparation of cycling egg extract (Shechter lab)

2011-11-15T19:44:24Z

moved Shechter Lab - preparation of cycling egg extract (extract that proceeds through multiple rounds of mitosis/interphase) to preparation of cycling egg extract (Shechter lab)

← Older revision	Revision as of 19:44, 15 November 2011
(No difference)

imported>Virgilio: moved Preparation of cycling egg extract (extract that proceeds through multiple rounds of mitosis/interphase) to Shechter Lab - preparation of cycling egg extract (extract that proceeds through multiple rounds of mitosis/interphase): added l...

2011-11-15T14:51:19Z

moved Preparation of cycling egg extract (extract that proceeds through multiple rounds of mitosis/interphase) to Shechter Lab - preparation of cycling egg extract (extract that proceeds through multiple rounds of mitosis/interphase): added l...

← Older revision	Revision as of 14:51, 15 November 2011
(No difference)

205.142.197.33: Created page with "Protocol submitted by VGP from David Shechter lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1810&tabId=0] Link to PDF: [http://www.shechterl..."

2011-11-11T20:11:04Z

Created page with "Protocol submitted by VGP from David Shechter lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1810&tabId=0] Link to PDF: [http://www.shechterl..."

New page

Protocol submitted by VGP from David Shechter lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1810&tabId=0]

Link to PDF: [http://www.shechterlab.org/wp-content/uploads/2009/04/Xenopus-Cycling-Egg-Extract.pdf]

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.