Richard Lab:Preparing electrocompetent cells

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Current revision (12:38, 15 September 2009) (view source)
(Day 2)
 
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====Day 2====
====Day 2====
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1. Fast cool the centrifuge with the correct rotor to 4°C.<br>
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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>  
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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]]
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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>  
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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===

Current revision

This protocol is for the preparation of electro-competent E. coli cells which are used for electroporation in the Richard Lab.

The consensus protocol should be consulted if deviating from the procedure outlined here.


Contents

Procedure

Liquid Nitrogen must be purchased from the Chemistry-Stockroom by 11:30AM.

Day 1

1. Autoclave the following

9x125ml flasks containing 50 mL LB Lennox or SOB
400 mL Millipore Water
100 mL 10% glycerol

2. While the flasks are still hot, combine the media flasks until you have four flasks containing 100ml and one flask containing 50ml.

3. Place the following in the refrigerator overnight:

Autoclaved water
Autoclaved Glycerol solution

4. Inoculate the 5OmL flask with NEB5 (DH5alpha) cells and grow overnight at 37°C and 240RPM.

Day 2

1. Fast cool the centrifuge with the correct rotor to 4°C.

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)

3. Pour the log phase culture into eight 50 mL centrifuge tubes.

4. Place the tubes on ice for 30 minutes.

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

5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.

6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water.

7. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.

8. Remove supernatant and gently resuspend pellets in 30ml ice-cold sterile water.

9. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.

10. Remove supernatant and gently resuspend pellets in 10ml cold 10% glycerol.

11. Transfer to 15 mL centrifuge tubes and hold on ice for 30 minutes.

12. Change the rotor inserts in the centrifuge to accomodate the 15ml tubes.

12. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.

13. Remove the supernatant and add 500 μl of 10% glycerol.

14. Pipet 100μl aliquots into micro-centrifuge tubes (on ice!!!).

15. Shock freeze cell suspensions using liquid nitrogen and store at -80°C.

Notes

  • The Ice-cold thing is really important
  • It is usually helpfull to have two people for the last steps of this protocol, that way one person can pipet 100μL of cell suspension into a tube, and the other person can dip the tube in liquid nitrogen. This saves you from having to keep all those little micro-centrifuge tubes on ice (Which is a real pain in the ass)


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