Richard Lab:Preparing electrocompetent cells: Difference between revisions
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::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 | 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> | |||
3. Place the following in the refrigerator overnight:<br> | 3. Place the following in the refrigerator overnight:<br> | ||
::Autoclaved water<br> | ::Autoclaved water<br> |
Revision as of 10:19, 15 September 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
Day 1
1. Autoclave the following
- 9x125ml flasks containing 50 mL LB Lennox or SOB
- 400 mL Millipore Water
- 100 mL 10% glycerol
- 9x125ml flasks containing 50 mL LB Lennox or SOB
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.
3. Place the following in the refrigerator overnight:
- Autoclaved water
- Autoclaved Glycerol solution
- Autoclaved water
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.
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. 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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