"

From 2013.igem.org

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
Tri-Na Citrate 300mM(Sub3)	10ml	1ml

Sub2: MC 10x 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