Richard Lab:Preparing electrocompetent cells

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(New page: <center> '''This protocol is for the typical electro-transformation of ''E. coli'' done in the Richard Lab. '''The consensus electroporation protocol should be cons...)
(Procedure)
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===Procedure===
===Procedure===
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Liquid Nitrogen must be purchased from the Chemistry-Stockroom by 11:30AM.
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<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.
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1. Inoculate 5mL LB Lennox medium and grow overnight at 37°C with rotation.<br>
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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)
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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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3. Autoclave the following
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3. Autoclave the following<br>
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200 mL LB Lennox or SOB
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::200 mL LB Lennox or SOB<br>
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400 mL Millipore Water
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::400 mL Millipore Water<br>
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50 mL 10% glycerol
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::50 mL 10% glycerol<br>
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4. Prechill the following:
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4. Prechill the following:<br>
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Autoclaved water
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::Autoclaved water<br>
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Autoclaved Glycerol solution
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::Autoclaved Glycerol solution<br>
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Four 50ml centrifuge tubes
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::Four 50ml centrifuge tubes<br>
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Four 15ml centrifuge tubes
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::Four 15ml centrifuge tubes<br>
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Twenty microcentrifuge tubes
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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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5. Fast cool the centrifuge with the correct rotor to 4°C  
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6. Pour the log phase culture into four 50 mL centrifuge tubes.<br>
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6. Pour the log phase culture into four 50 mL centrifuge tubes.  
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7. Place the tubes on ice for 30 minutes.<br>  
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7. Place the tubes on ice for 30 minutes.  
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<center>'''For the following steps it is important to keep cells ice-cold'''</center>
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-
 
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8. Centrifuge for 10 mins at 2000g at 4°C
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9. Remove supernatant and gently resuspend pellets with 40ml ice-cold sterile water.
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11. Centrifuge for 15 mins at 2000g at 4°C
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12. Remove supernatant and gently resuspend pellets in 20ml cold sterile water.
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13. Hold on ice for 30 minutes
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14. Centrifuge for 15 mins at 2000g at 4°C
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15. Remove supernatant and gently resuspend pellets in 10ml cold 10% glycerol. 
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16. Transfer to 15 mL centrifuge tubes and hold on ice for 30 minutes
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17. Centrifuge for 15 mins at 1500g at 4°C
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18. Remove the supernatant and add 500 μl of 10% glycerol
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19. Aliquot 100 μL per tube (tubes on ice)
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20. Shock freeze cell suspensions using liquid nitrogen and store at -80°C
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 +
<center>'''For the following steps it is important to keep cells ice-cold'''</center><br>
 +
8. Centrifuge for 10 mins at 2000g at 4°C. <br>
 +
9. Remove supernatant and gently resuspend pellets with 40ml ice-cold sterile water. <br>
 +
11. Centrifuge for 15 mins at 2000g at 4°C.<br>
 +
12. Remove supernatant and gently resuspend pellets in 20ml cold sterile water.<br>
 +
13. Hold on ice for 30 minutes.<br>
 +
14. Centrifuge for 15 mins at 2000g at 4°C.<br>
 +
15. Remove supernatant and gently resuspend pellets in 10ml cold 10% glycerol.<br> 
 +
16. Transfer to 15 mL centrifuge tubes and hold on ice for 30 minutes.<br>
 +
17. Centrifuge for 15 mins at 1500g at 4°C.<br>
 +
18. Remove the supernatant and add 500 μl of 10% glycerol.<br>
 +
19. Aliquot 100 μL per tube (tubes on ice).<br>
 +
20. Shock freeze cell suspensions using liquid nitrogen and store at -80°C.<br>
===Notes===
===Notes===

Revision as of 15:42, 23 June 2009

This protocol is for the typical electro-transformation of E. coli done in the Richard Lab.

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


Procedure

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

1. Inoculate 5mL LB Lennox medium and grow overnight at 37°C with rotation.
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)
3. Autoclave the following

200 mL LB Lennox or SOB
400 mL Millipore Water
50 mL 10% glycerol

4. Prechill the following:

Autoclaved water
Autoclaved Glycerol solution
Four 50ml centrifuge tubes
Four 15ml centrifuge tubes
Twenty microcentrifuge tubes

5. Fast cool the centrifuge with the correct rotor to 4°C.
6. Pour the log phase culture into four 50 mL centrifuge tubes.
7. Place the tubes on ice for 30 minutes.

For the following steps it is important to keep cells ice-cold

8. Centrifuge for 10 mins at 2000g at 4°C.
9. Remove supernatant and gently resuspend pellets with 40ml ice-cold sterile water.
11. Centrifuge for 15 mins at 2000g at 4°C.
12. Remove supernatant and gently resuspend pellets in 20ml cold sterile water.
13. Hold on ice for 30 minutes.
14. Centrifuge for 15 mins at 2000g at 4°C.
15. Remove supernatant and gently resuspend pellets in 10ml cold 10% glycerol.
16. Transfer to 15 mL centrifuge tubes and hold on ice for 30 minutes.
17. Centrifuge for 15 mins at 1500g at 4°C.
18. Remove the supernatant and add 500 μl of 10% glycerol.
19. Aliquot 100 μL per tube (tubes on ice).
20. Shock freeze cell suspensions using liquid nitrogen and store at -80°C.

Notes

  • 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).
  • Other protocols (specificially the Knight Protocol) were used extensively in the development of this protocol and have much more accessory information than this protocol contains. Feel free to access these protocols from the consensus protocol page


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