Richard Lab:Preparing electrocompetent cells - Revision history

Michael A. Speer: /* Day 2 */

2009-09-15T17:38:36Z

Day 2

Michael A. Speer at 17:37, 15 September 2009

2009-09-15T17:37:47Z

←Older revision		Revision as of 17:37, 15 September 2009
Line 10:		Line 10:
	::9x125ml flasks containing 50 mL LB Lennox or SOB<br>		::9x125ml flasks containing 50 mL LB Lennox or SOB<br>
	::400 mL Millipore Water<br>		::400 mL Millipore Water<br>
-	::100 mL 10% glycerol<br>	+	::100 mL 10% glycerol<br><br>
-	2. While the flasks are still hot, combine the media flasks until you have four flasks containing 100ml and one flask containing 50ml.<br>	+	2. While the flasks are still hot, combine the media flasks until you have four flasks containing 100ml and one flask containing 50ml.<br><br>
	3. Place the following in the refrigerator overnight:<br>		3. Place the following in the refrigerator overnight:<br>
	::Autoclaved water<br>		::Autoclaved water<br>
-	::Autoclaved Glycerol solution<br>	+	::Autoclaved Glycerol solution<br><br>
	4. Inoculate the 5OmL flask with NEB5 (DH5alpha) cells and grow overnight at 37°C and 240RPM.<br>		4. Inoculate the 5OmL flask with NEB5 (DH5alpha) cells and grow overnight at 37°C and 240RPM.<br>

Michael A. Speer: /* Day 2 */

2009-09-15T17:36:54Z

Day 2

←Older revision		Revision as of 17:36, 15 September 2009
Line 23:		Line 23:
	4. Place the tubes on ice for 30 minutes.<br>		4. Place the tubes on ice for 30 minutes.<br>
	<center>'''For the following steps it is important to keep cells ice-cold. You will also have to alternate the tubes because the centrifuge can only hold 4 tubes at a time. A diagram on how to do this can be found to the right'''</center><br>		<center>'''For the following steps it is important to keep cells ice-cold. You will also have to alternate the tubes because the centrifuge can only hold 4 tubes at a time. A diagram on how to do this can be found to the right'''</center><br>
-	[[Image:Centrifugation_schedule.jpg\|~~150px~~\|right]]	+	[[Image:Centrifugation_schedule.jpg\|200px\|right]]
	5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C. <br>		5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C. <br>
	6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water. <br>		6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water. <br>

Michael A. Speer: /* Day 2 */

2009-09-15T17:36:20Z

Day 2

←Older revision		Revision as of 17:36, 15 September 2009
Line 23:		Line 23:
	4. Place the tubes on ice for 30 minutes.<br>		4. Place the tubes on ice for 30 minutes.<br>
	<center>'''For the following steps it is important to keep cells ice-cold. You will also have to alternate the tubes because the centrifuge can only hold 4 tubes at a time. A diagram on how to do this can be found to the right'''</center><br>		<center>'''For the following steps it is important to keep cells ice-cold. You will also have to alternate the tubes because the centrifuge can only hold 4 tubes at a time. A diagram on how to do this can be found to the right'''</center><br>
-	[[Image:Centrifugation_schedule.jpg\|~~50px~~\|right]]	+	[[Image:Centrifugation_schedule.jpg\|150px\|right]]
	5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C. <br>		5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C. <br>
	6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water. <br>		6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water. <br>

Michael A. Speer: /* Day 2 */

2009-09-15T17:36:00Z

Day 2

←Older revision		Revision as of 17:36, 15 September 2009
Line 23:		Line 23:
	4. Place the tubes on ice for 30 minutes.<br>		4. Place the tubes on ice for 30 minutes.<br>
	<center>'''For the following steps it is important to keep cells ice-cold. You will also have to alternate the tubes because the centrifuge can only hold 4 tubes at a time. A diagram on how to do this can be found to the right'''</center><br>		<center>'''For the following steps it is important to keep cells ice-cold. You will also have to alternate the tubes because the centrifuge can only hold 4 tubes at a time. A diagram on how to do this can be found to the right'''</center><br>
-	[[Image:Centrifugation_schedule.jpg\|~~300px~~\|right]]	+	[[Image:Centrifugation_schedule.jpg\|50px\|right]]
	5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C. <br>		5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C. <br>
	6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water. <br>		6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water. <br>

Michael A. Speer: /* Day 2 */

2009-09-15T17:35:44Z

Day 2

←Older revision		Revision as of 17:35, 15 September 2009
Line 23:		Line 23:
	4. Place the tubes on ice for 30 minutes.<br>		4. Place the tubes on ice for 30 minutes.<br>
	<center>'''For the following steps it is important to keep cells ice-cold. You will also have to alternate the tubes because the centrifuge can only hold 4 tubes at a time. A diagram on how to do this can be found to the right'''</center><br>		<center>'''For the following steps it is important to keep cells ice-cold. You will also have to alternate the tubes because the centrifuge can only hold 4 tubes at a time. A diagram on how to do this can be found to the right'''</center><br>
-	[[Image:Centrifugation_schedule.~~JPG~~\|300px\|right]]	+	[[Image:Centrifugation_schedule.jpg\|300px\|right]]
	5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C. <br>		5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C. <br>
	6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water. <br>		6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water. <br>

Michael A. Speer: /* Day 2 */

2009-09-15T17:35:14Z

Day 2

←Older revision		Revision as of 17:35, 15 September 2009
Line 23:		Line 23:
	4. Place the tubes on ice for 30 minutes.<br>		4. Place the tubes on ice for 30 minutes.<br>
	<center>'''For the following steps it is important to keep cells ice-cold. You will also have to alternate the tubes because the centrifuge can only hold 4 tubes at a time. A diagram on how to do this can be found to the right'''</center><br>		<center>'''For the following steps it is important to keep cells ice-cold. You will also have to alternate the tubes because the centrifuge can only hold 4 tubes at a time. A diagram on how to do this can be found to the right'''</center><br>
		+	[[Image:Centrifugation_schedule.JPG\|300px\|right]]
	5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C. <br>		5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C. <br>
	6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water. <br>		6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water. <br>

Michael A. Speer: /* Day 2 */

2009-09-15T17:23:50Z

Day 2

←Older revision		Revision as of 17:23, 15 September 2009
Line 29:		Line 29:
	9. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.<br>		9. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.<br>
	10. Remove supernatant and gently resuspend pellets in 10ml cold 10% glycerol.<br>		10. Remove supernatant and gently resuspend pellets in 10ml cold 10% glycerol.<br>
-	11. Transfer to 15 mL centrifuge tubes and hold on ice for 30 minutes.<br>	+	11. Transfer to 15 mL centrifuge tubes and hold on ice for 30 minutes.<br>
		+	12. Change the rotor inserts in the centrifuge to accomodate the 15ml tubes.<br>
	12. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.<br>		12. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.<br>
	13. Remove the supernatant and add 500 μl of 10% glycerol.<br>		13. Remove the supernatant and add 500 μl of 10% glycerol.<br>

Michael A. Speer: /* Day 1 */

2009-09-15T17:20:29Z

Day 1

←Older revision		Revision as of 17:20, 15 September 2009
Line 11:		Line 11:
	::400 mL Millipore Water<br>		::400 mL Millipore Water<br>
	::100 mL 10% glycerol<br>		::100 mL 10% glycerol<br>
-	2. While the flasks are still hot, ~~pair and~~ combine ~~eight of~~ the flasks until you have four flasks containing 100ml and one flask containing 50ml ~~of media~~.<br>	+	2. While the flasks are still hot, combine the media flasks until you have four flasks containing 100ml and one flask containing 50ml.<br>
	3. Place the following in the refrigerator overnight:<br>		3. Place the following in the refrigerator overnight:<br>
	::Autoclaved water<br>		::Autoclaved water<br>

Michael A. Speer: /* Day 1 */

2009-09-15T17:19:38Z

Day 1

←Older revision		Revision as of 17:19, 15 September 2009
Line 11:		Line 11:
	::400 mL Millipore Water<br>		::400 mL Millipore Water<br>
	::100 mL 10% glycerol<br>		::100 mL 10% glycerol<br>
-	2. While the flasks are still hot, pair and combine eight of the flasks	+	2. While the flasks are still hot, pair and combine eight of the flasks until you have four flasks containing 100ml and one flask containing 50ml of media.<br>
-	:until you have four flasks containing 100ml and one flask containing 50ml of media.<br>	+
	3. Place the following in the refrigerator overnight:<br>		3. Place the following in the refrigerator overnight:<br>
	::Autoclaved water<br>		::Autoclaved water<br>

←Older revision		Revision as of 17:38, 15 September 2009
Line 18:		Line 18:

	====Day 2====		====Day 2====
-	1. Fast cool the centrifuge with the correct rotor to 4°C.<br>	+	1. Fast cool the centrifuge with the correct rotor to 4°C.<br><br>
-	2. Add the 5mL of the overnight culture to each (there are 4) flask containing 100ml of LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. (approximately 3 hours)<br>	+	2. Add the 5mL of the overnight culture to each (there are 4) flask containing 100ml of LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. (approximately 3 hours)<br><br>
-	3. Pour the log phase culture into eight 50 mL centrifuge tubes.<br>	+	3. Pour the log phase culture into eight 50 mL centrifuge tubes.<br><br>
-	4. Place the tubes on ice for 30 minutes.<br>	+	4. Place the tubes on ice for 30 minutes.<br><br>
	<center>'''For the following steps it is important to keep cells ice-cold. You will also have to alternate the tubes because the centrifuge can only hold 4 tubes at a time. A diagram on how to do this can be found to the right'''</center><br>		<center>'''For the following steps it is important to keep cells ice-cold. You will also have to alternate the tubes because the centrifuge can only hold 4 tubes at a time. A diagram on how to do this can be found to the right'''</center><br>
	[[Image:Centrifugation_schedule.jpg\|200px\|right]]		[[Image:Centrifugation_schedule.jpg\|200px\|right]]
-	5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C. <br>	+	5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C. <br><br>
-	6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water. <br>	+	6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water. <br><br>
-	7. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.<br>	+	7. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.<br><br>
-	8. Remove supernatant and gently resuspend pellets in 30ml ice-cold sterile water.<br>	+	8. Remove supernatant and gently resuspend pellets in 30ml ice-cold sterile water.<br><br>
-	9. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.<br>	+	9. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.<br><br>
-	10. Remove supernatant and gently resuspend pellets in 10ml cold 10% glycerol.<br>	+	10. Remove supernatant and gently resuspend pellets in 10ml cold 10% glycerol.<br><br>
-	11. Transfer to 15 mL centrifuge tubes and hold on ice for 30 minutes.<br>	+	11. Transfer to 15 mL centrifuge tubes and hold on ice for 30 minutes.<br><br>
-	12. Change the rotor inserts in the centrifuge to accomodate the 15ml tubes.<br>	+	12. Change the rotor inserts in the centrifuge to accomodate the 15ml tubes.<br><br>
-	12. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.<br>	+	12. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.<br><br>
-	13. Remove the supernatant and add 500 μl of 10% glycerol.<br>	+	13. Remove the supernatant and add 500 μl of 10% glycerol.<br><br>
-	14. Pipet 100μl aliquots into micro-centrifuge tubes (on ice!!!).<br>	+	14. Pipet 100μl aliquots into micro-centrifuge tubes (on ice!!!).<br><br>
-	15. Shock freeze cell suspensions using liquid nitrogen and store at -80°C.<br>	+	15. Shock freeze cell suspensions using liquid nitrogen and store at -80°C.<br><br>

	===Notes===		===Notes===