Team:UCL London/Logistics

CaCl2 Competent Bacteria - Generating and Transforming

Generating

Before Starting;

Pour minimal media plates. 5x M9 salts etc overleaf. Prepare 100ml LB per strain. Prepare 50ml ice cold 0.1M CaCl2 / 15% glycerol per strain Pre-chill eppendorf tubes

Day 1

1 Streak cells on minimal agar plate. Incubate 37C overnight.

Day 2

2 Pick a colony into 5ml LB + 100μl 1M MgSO4 Incubate 37C in shaker overnight.

Day 3

3 Inoculate 100ml LB in pre-warmed conical with 1ml of the 5ml O/N culture from Day 2.

4 Incubate 2hrs in 37C shaker until the cells at early log phase of growth curve (A600 ~ 0.3)

5 Transfer to chilled, sterile 50ml Falcon tube and incubate on ice 10min

6 Cf 3300g 5min in benchtop RmT. Cf.

7 Resus in 10ml ice cold 0.1M CaCl2 / 15% glycerol and incubate on ice 30min

8 Cf 3300g 5min 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 -70C.

Solutions

5x M9 salts in 500ml dH20.

Na2HPO4 32g KH2PO4 7.5g NaCl 1.25g NH4Cl 2.5g

Minimal media plates for competence

In 50 ml Falcon;

39ml melted Bacteriological Agar solution (≤50C) 10ml 5x M9 salt solution 1ml 20% (w/v) D-glucose 50μl 2mg ml-1 thiamine 5μl 1M CaCl2 100μl 1M MgSO4

0.1M CaCl2 / 15% glycerol

In 50 ml Falcon;

5ml 1M CaCl2 7.5ml 100% glycerol

Transforming

Before Starting

Prepare 5ml LB per strain. Prepare appropriately selective nutrient agar plates

Day 1

1 Add 1-5 μg of ice cold DNA to 100 μl of competent cells on ice. Incubating for 45 min.

2 Heat shock cells by placing tubes in 37C water bath for 10min.

3 Place cells on ice 2 min

4 Add cells to 5ml LB (in 15ml or 50ml tube). Shake for 1 hour at 37C.

5 Plate at least 100μl onto selective nutrient agar plates.

Day 2

6 Pick a colony into 2ml selective LB. Incubate 37C in shaker overnight.