Team:Groningen/protocols/Transformation

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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
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 H2O and mix. When everything is dissolved add H2O till 100ml. Filter sterilize the complete mixture and store at -20°C

Sub3: Tri-Na Citrate 300mM
compound amount
Tri-Na Citrate 0.88g
H2O 10 ml
Wrap in aluminium foil and store at -20°C
Sub4: Ferric NH4 citrate
compound amount
Ferric NH4 0.22g
H2O 10 ml
Wrap in aluminium foil and store at -20°C