Team:Groningen/protocols/Transformation

B. subtilis transformation

The Losick protocol is used

Day 0: Streak out the Bacillus strain of use and plate this on an LB agar plate o/n at 37°C.

Transformation (D-Day):
1. Pick up a nice big colony and drop it in 2ml of completed MC (1x) (see sub1).
2. Grow at 37 °C for 5 hours (longer if the culture is not really turbid).
3. Mix 400µl of culture with DNA* in a fresh tube (i.e. 15ml tubes loosely closed – aeration)
4. Grow the cells at 37°C for an additional 2 hours .
5. Spread the complete 400µl reaction mix on selective antibiotic plates, and incubate at 37°C overnight.

(*usually 1µl. Then 10µl of Quiagen plasmid miniprep or <1µl of chromosomal prep)

Sub1: Competence medium (MC completed)

compound	amount	treatment
H2O?	1.8ml
10x MC (Sub2)	200µl	filter sterilized
MgSO4	6.7µl	autoclaved
Tryptophan 1%	10µl	filter sterilized (stored in aluminium foil)

Sub2: MC 10x

compound	amount 10ml	amount 100ml
K2HPO4	1.40g	14.04g
KH2PO4	0.52g	5.24g
Glucose	2g	20g
Tri-Na Citrate 300mM (Sub3)	1ml	10ml
Ferric NH4 citrate (Sub4)	0.1ml	1ml
Casein Hydrolysate	0.1g	1g
Potassium Glutamate	0.2g	2g

The complete mixture should be dissolved in 100ml. First add 50ml H₂O and mix. When everything is dissolved add H₂O till 100ml. Filter sterilize the complete mixture and store at -20°C

Sub3: Tri-Na Citrate 300mM

compound	amount
Tri-Na Citrate	0.88g
H2O	10ml

Wrap in aluminium foil and store at -20°C

Sub4: Ferric NH4 citrate

compound	amount
Ferric NH4	0.22g
H2O	10ml

Wrap in aluminium foil and store at -20°C