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

for	100 ml	10 ml
K2H PO4	14,036g	1,4036g
KH2 PO4	5,239g	0,5239g
Glucose	20g	2g
Tri-Na Citrate 300mM(Sub3)	10ml	1ml
Ferric NH4 citrate(Sub4)	1ml	0,1ml
Casein Hydrolysate	1g	0,1g
Potassium Glutamate	2g	0,2g

Add 50ml H2O, Mix, add H2O till 100ml, Filter sterilize and freeze at -20 °C

Sub3: Tri-Na Citrate 300mM

Tri-Na Citrate	0,8823g
H2O	10ml

Wrap in aluminium

Sub4: Ferric NH4 citrate

Ferric NH4 citrate	0,22g
H2O	10ml

Wrap in aluminium