Competent agro prep for electroporation

day 1

1. Start 75 mL overnight cultures of agro (strain GV3101 C58C1 Rifr pMP90 Gmr, Koncz & Schell) in YEP in 250 mL baffle flasks.

2. Grow at 28 °C, shaking.

3. Prepare:

~ 3.5 L sterile H2O chilled to 4 °C.

10 % glycerol, autoclaved chilled to 4 °C.

Centrifuge bottles (4), autoclaved.

day 2

4. From each overnight culture, innoculate 1 L YEP in 2 L baffle flasks (1:200).

5. Grow overnight at 28 °C shaking.

day 3

6. Allow 1 L cultures to grow to OD595 = 1.5.

7. Distribute cultures into centrifuge bottles + pellet cells. 15 min, 5000 rpm.

8. Resuspend cells in cold sterile H2O (2 L total).

9. Pellet cells. 15 min, 5000 rpm.

10. Resuspend cells in cold sterile H2O (1 L total).

11. Pellet cells. 15 min, 5000 rpm.

12. Resuspend cells in 2 20 mL aliquots of cold 10% glycerol + transfer to Oakridge tubes.

13. Pellet cells. 15 min, 6000 rpm.

14. Resuspend each pellet in 1 mL 10% glycerol (cold).

15. Aliquot into 1.5 mL eppendorf on ice, 80 µL each + freeze in EtOH/dry ice bath.

16. Store at –80°C.

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References:

Koncz C, Schell J (1986) Mol. Gen. Genet. 204:383-396

Solutions

YEP medium (1 liter)

10 g Bacto peptone

10 g yeast extract

5 g NaCl