Monday, November 10, 2003

Description
Making electrocompetent Agrobacterium

Procedure
1. Start a 5ml culture (LB plus appropriate selection - for GV2260 this is Rif25, Strep50 and Carb50) from a freshly streaked plate. Incubate at 28C overnight (approx. 16 hours).
2. The following morning inoculate 100 ml of media (LB plus appropriate selection) with the overnight culture. Incubate at 28C until culture reaches an OD600 of 0.8-1.0.
3. Pellet cells by spinning in a chilled (2C) Sorvall GSA rotor for 10 minutes at 4000 rpm.
4. Pour off supernatant and resuspend pellet in 5 ml chilled sterile water by pipetting up and down. After pellet is resuspended, add 100ml cold, sterile water.
5. Spin again (4000 rpm, 2C) for 15 minutes.
6. Pour off supernatant and resuspend pellet in 100ml cold, sterile water.
7. Spin again (4000 rpm, 2C) for 20 minutes.
8. Pour off supernatant and resuspend pellet in 40ml cold, sterile 10% glycerol.
9. Pellet cells by spinning in chilled (2C) Sorvall SS-34 rotor (in Corning 50ml polypropylene tubes) for 15 minutes at 3000 rpm. NOTE: The 50ml tubes will fit in rotor but cannot be accommodated by the lid - therefore, make sure that the rotor is bolted into the centrifuge.
10. Quickly pour off supernatant - pellet will be very loose - and resuspend in an equal volume of 20% cold, sterile glycerol. (Generally this is between 0.5-1.0ml).
11. Cells can be used immediately or aliquoted and frozen at -80C to be used at a later time.

Transformation electrocompetent Agrobacterium:

1. Use 100-150ul of competent cells with 500ng-1ug plasmid.
2. Electroporate cells at 2.47 V, 2000 ohms, 25 uF.
3. After shocking cells add 1 ml LB and shake for 2 hours at 28C.
4. Plate cells on LBA plus appropriate selection

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