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