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Team:UCL London/From the lab/Protocols
From 2009.igem.org
Competent Bacteria:
- Materials:
- 5×M9 salts in 500ml dH2O:
- Na2HPO4--- 32g
- KH2PO4 --- 7.5g
- NaCl --- 1.25g
- NH4Cl --- 2.5g
- Minimal media: ( In 50ml Falcon )
- Melted bacteriological agar solution (< 50°C) --- 39ml
- 5×M9 salt solution --- 10ml
- 20% (w/v) D-glucose --- 1ml
- 1M CaCl2 --- 5µl
- 1M MgSO4 --- 100µl
- 0.1M CaCl2 / 15% glycerol: (In 59 ml Falcon)
- 1M CaCl2 --- 5ml
- 100% glycerol --- 7.5ml
- Preparation:
- Pour minimal media plates.
- 5x M9 salts.
- Prepare 100ml LB per strain.
- Prepare 50ml ice cold 0.1M CaCl2 / 15% glycerol per strain
- Pre-chill eppendorf tubes
- Method:
- Streak cells on minimal agar plate. Incubate 37°C overnight.
- Pick a colony into 5 ml LB + 100µl 1M MgSO4. Incubate 37°C overnight.
- Inoculate 100ml LB in pre-warmed conical with 1ml of the 5ml O/N culture from Step 2.
- Incubate 2 hrs in 37°C shaker until the cells at early log phase of growth curve (A600 ~ 0.3).
- Transfer to chilled, sterile two 50ml Falcon tubes and incubate on ice for 10 min.
- Cf 3300g 5 min in bechtop RmT. Cf.
- Resus in 10ml ice cold 0.1M CaCl2 / 15% glycerol and incubate on ice 30 min.
- Cf 3300g 5 min in benchtop RmT. Cf.
- Resus in 1ml ice cold 0.1M CaCl2 / 15% glycerol. Transfer 100µl aliquots to pre-chilled, pre-labelled eppendorf tubes. Store -80°C.
