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B. subtilis transformation
Losick protocol

-1 Day: Streak out strain and incubate 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 (more if culture is not really turbid).
3. Mix 400 µl of culture with DNA* in fresh tube (i.e. 15 ml tubes loosely closed – aeration) (*usually 1 µl. Then 10 µl of Quiagen plasmid miniprep or <1 µl of chromosal prep)
4. Grow for an additional 2 hours at 37 °C.
5. Plate all on selective antibiotic plates, and incubates at 37°C o/n.

Sub1: Copmpetence medium (MC completed)

H20 1,8 ml
10x MC (sub2) 200 ul filter sterilize
MgSO4 6,7 ul autoclave sterilize
Tryptophan 1% 10 ul filter steralize, wrap in Al

Sub2: MC 10x

for 100 ml 10 ml
K2H PO4 14,036g 1,4036g
KH2 PO$ 5,239g 0,5239g
Glucose 20g 2g

Sub2: MC 10x 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 aluminum

Sub4: Ferric NH4 citrate Ferric NH4 citrate 0,22g H2O 10ml Wrap in aluminum