July 22nd

We have 4 tubes from last year. These strains are frozen in glycerol.

PNZ8901 plasmid in E.coli MC1061 Strain
- Chloroamphenicol resistant
- "sure" vector
- does not integrate

B. subtilis strain 1A1
- No resistance
- Same as strain 168 but tryptophan deficient

Checking antibiotic resistance

Purpose : Grow each of them on a plate to test their antibiotic resistance

Protocol

Take one colony from each of the plates grown from the tubes yesterday and plated them again on LA without antibiotics

1/ Purpose Check antibiotic resistance of our different strains

Strain	Plasmid	Extracted from	Antibiotic use	Concentration of antibiotic
DH5alpha	PP182	tube	Amp	100
MC1061	NONE	tube	Cm	35
MC1061	PNZ8901	tube	Cm	35
DH5alpha	PP182	plate	Amp	100
MC1061	NONE	plate	Cm	35
MC1061	PNZ8901	plate	Cm	35

2/ Purpose : Find the right concentration of antibiotic so that B. subtilis survive

- Grow 15uL of B. 1A1 (frozen tube) in 5mL of LB

- Incubate at 37°C

3/ Purpose : Grow plasmids in TOP10, transformation

4 plasmids :

1 control : PUC19

- Add 20uL of TOP10 competent cells and 0.5uL of Plasmid in an eppendorf

- 30min on ice

- Heat shock : 60s at 42°C

- 2min on ice

- Add 89.5uL of SOC

- 60min at 37°C

- Put the mix on plate with ampicillin resistance

- Incubate at 37°C