Jan 06, 2025
Preparation of Chemically competent Agrbacterium cells
This protocol is a draft, published without a DOI.
- 1CSJMU, Kanpur
Protocol Citation: Velu Mani Selvaraj 2025. Preparation of Chemically competent Agrbacterium cells. protocols.io https://protocols.io/view/preparation-of-chemically-competent-agrbacterium-c-dw6u7hew
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: January 06, 2025
Last Modified: January 06, 2025
Protocol Integer ID: 117684
Abstract
Preparation of chemically competent Agrobacterium cells using calcium chloride
Materials
Composition of YEM medium (1 Lt)
✔️Mannitol 10 g
✔️Yeast extract 0.4 g
✔️NaCl 0.1 g
✔️MgSO4, 7H2O 0.02 g
✔️0.5% K2HPO4 10 ml (prepare separately with autoclaved water, filter sterilize and add at the time of pouring plate)
✔️Agar 15g
Streak Agrobacterium strain on YEM plate containing rifampicin (stock 20 mg/ml; dissolve in methanol). Grow at 28 C for 48 hrs.
Inoculate single colony in 5 ml YEM + Rif. And grow at 28 C for 12-16 hours
Inoculate 2 ml culture to 50 ml YEM + Rif in a 250 ml flask and shake vigorously (250 rpm) at 28 C until culture grows to an OD600 of 0.5-1.0 (5-6 hrs)
Chill the culture on ice. Centrifuge cell suspension at 3000 xg for 5 min at 4 C.
Resuspend cells in 1 ml of 20 mM CaCl2 (0.03 g CaCl2 in 10 ml autoclaved water, filter). Centrifuge at 300 xg for 5 min at 4 C. Discard supernatant.
Resuspend pellet in 1 ml of 20 mM CaCl2. Dispense 0.1 ml of aliquots into prechilled EP tubes. Freeze in liquid nitrogen and store at -80 C.