Richard Lab:Preparing electrocompetent cells

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(Procedure)
Current revision (12:38, 15 September 2009) (view source)
(Day 2)
 
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<center>
<center>
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'''This protocol is for the typical electro-transformation of ''E. coli'' done in the Richard Lab.   
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'''This protocol is for the preparation of electro-competent ''E. coli'' cells which are used for electroporation in the Richard Lab.   
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'''The [[Electroporation | consensus electroporation protocol]] should be consulted if deviating from the procedure outlined here.'''</center>
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'''The [[Electrocompetent_Cells | consensus protocol]] should be consulted if deviating from the procedure outlined here.'''</center>
===Procedure===
===Procedure===
<center>'''Liquid Nitrogen must be purchased from the Chemistry-Stockroom by 11:30AM.'''</center>
<center>'''Liquid Nitrogen must be purchased from the Chemistry-Stockroom by 11:30AM.'''</center>
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1. Inoculate 5mL LB Lennox medium and grow overnight at 37°C with rotation.<br>
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====Day 1====
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2. Add the 5mL overnight culture to 200mL LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. (3 hours)<br>
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1. Autoclave the following<br>
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3. Autoclave the following<br>
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::9x125ml flasks containing 50 mL LB Lennox or SOB<br>
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::200 mL LB Lennox or SOB<br>
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::400 mL Millipore Water<br>
::400 mL Millipore Water<br>
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::50 mL 10% glycerol<br>
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::100 mL 10% glycerol<br><br>
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4. Prechill the following:<br>
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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> 
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3. Place the following in the refrigerator overnight:<br>
::Autoclaved water<br>
::Autoclaved water<br>
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::Autoclaved Glycerol solution<br>
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::Autoclaved Glycerol solution<br><br>
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::Four 50ml centrifuge tubes<br>
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4. Inoculate the 5OmL flask with NEB5 (DH5alpha) cells and grow overnight at 37°C and 240RPM.<br>
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::Four 15ml centrifuge tubes<br>
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::Twenty microcentrifuge tubes<br>
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5. Fast cool the centrifuge with the correct rotor to 4°C.<br>
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6. Pour the log phase culture into four 50 mL centrifuge tubes.<br>
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7. Place the tubes on ice for 30 minutes.<br>  
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<center>'''For the following steps it is important to keep cells ice-cold'''</center><br>
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====Day 2====
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1. Fast cool the centrifuge with the correct rotor to 4°C.<br><br>
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8. Centrifuge for 10 mins at 2000g at 4°C. <br>
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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>
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9. Remove supernatant and gently resuspend pellets with 40ml ice-cold sterile water. <br>
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3. Pour the log phase culture into eight 50 mL centrifuge tubes.<br><br>
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11. Centrifuge for 15 mins at 2000g at 4°C.<br>  
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4. Place the tubes on ice for 30 minutes.<br><br>
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12. Remove supernatant and gently resuspend pellets in 20ml cold sterile water.<br>  
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<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>
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13. Hold on ice for 30 minutes.<br>  
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[[Image:Centrifugation_schedule.jpg|200px|right]]
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14. Centrifuge for 15 mins at 2000g at 4°C.<br>  
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5. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C. <br><br>
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15. Remove supernatant and gently resuspend pellets in 10ml cold 10% glycerol.<br>   
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6. Remove supernatant and gently resuspend pellets with 30ml ice-cold sterile water. <br><br>
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16. Transfer to 15 mL centrifuge tubes and hold on ice for 30 minutes.<br>  
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7. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.<br><br>  
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17. Centrifuge for 15 mins at 1500g at 4°C.<br>
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8. Remove supernatant and gently resuspend pellets in 30ml ice-cold sterile water.<br><br>  
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18. Remove the supernatant and add 500 μl of 10% glycerol.<br>  
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9. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.<br><br>  
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19. Aliquot 100 μL per tube (tubes on ice).<br>  
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10. Remove supernatant and gently resuspend pellets in 10ml cold 10% glycerol.<br><br>   
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20. Shock freeze cell suspensions using liquid nitrogen and store at -80°C.<br>
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11. Transfer to 15 mL centrifuge tubes and hold on ice for 30 minutes.<br><br>
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12. Change the rotor inserts in the centrifuge to accomodate the 15ml tubes.<br><br>
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12. Centrifuge for 15 mins at 2000g (3500 RPM) at 4°C.<br><br>
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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><br>  
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15. Shock freeze cell suspensions using liquid nitrogen and store at -80°C.<br><br>
===Notes===
===Notes===
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* When choosing the # of vials and cuvettes, be sure to include 2 vials for two negative controls (one with no DNA added, and another with only cut vector added).
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* The Ice-cold thing is really important
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*Other protocols (specificially the [[Knight:Electroporation|Knight Protocol]]) were used extensively in the development of this protocol and have much more accessory information than this protocol containsFeel free to access these protocols from the [[Electroporation|consensus protocol page]]
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* 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 nitrogenThis saves you from having to keep all those little micro-centrifuge tubes on ice (Which is a real pain in the ass)
[[Category:Protocol]]
[[Category:Protocol]]

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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