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
MQ water	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 milliQ water and mix. When everything is dissolved add MQ water till 100ml. Filter sterilize the complete mixture and store at -20°C

Sub3: Tri-Na Citrate 300mM

compound	amount
Tri-Na Citrate	0.88g
MQ water	10ml

Wrap in aluminium foil and store at -20°C

Sub4: Ferric NH4 citrate

compound	amount
Ferric NH4	0.22g
MQ water	10ml

Wrap in aluminium foil and store at -20°C