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

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

Sub1: Competence medium (MC completed)

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

Sub2: MC 10x

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

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

Sub3: Tri-Na Citrate 300 mM

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

Wrap in aluminium foil and store at -20°C

Sub4: Ferric NH4 citrate

compound	amount
Ferric NH4	0.22 g
MQ water	10 ml

Wrap in aluminium foil and store at -20°C