UCL London/Protocol/Competent Bacteria

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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:

1. Streak cells on minimal agar plate. Incubate 37°C overnight.
2. Pick a colony into 5 ml LB + 100µl 1M MgSO4. Incubate 37°C overnight.
3. Inoculate 100ml LB in pre-warmed conical with 1ml of the 5ml O/N culture from Step 2.
4. Incubate 2 hrs in 37°C shaker until the cells at early log phase of growth curve (A600 ~ 0.3).
5. Transfer to chilled, sterile two 50ml Falcon tubes and incubate on ice for 10 min.
6. Cf 3300g 5 min in bechtop RmT. Cf.
7. Resus in 10ml ice cold 0.1M CaCl2 / 15% glycerol and incubate on ice 30 min.
8. Cf 3300g 5 min in benchtop RmT. Cf.
9. Resus in 1ml ice cold 0.1M CaCl2 / 15% glycerol. Transfer 100µl aliquots to pre-chilled, pre-labelled eppendorf tubes. Store -80°C.