Richard Lab:Preparing electrocompetent cells

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(Notes)
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===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 centrifuge tubes on ice (Which is a real pain in the ass)
[[Category:Protocol]]
[[Category:Protocol]]

Revision as of 15:48, 23 June 2009

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.


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

  • 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 centrifuge tubes on ice (Which is a real pain in the ass)


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