UCL London/Protocol/Competent Bacteria

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.