Team:Groningen/protocols

From 2013.igem.org

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<td>10ml</td>
<td>10ml</td>
<td>1ml</td>
<td>1ml</td>
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</tr></table>
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</tr>
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<tr>
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<td>Ferric NH4 citrate(Sub4)</td>
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<td>1ml</td>
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<td>0,1ml</td>
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</tr>
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<tr>
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<td>Casein Hydrolysate</td>
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<td>1g</td>
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<td>0,1g</td>
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</tr>
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<tr>
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<td>Potassium Glutamate</td>
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<td>2g</td>
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<td>0,2g</td>
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</tr>
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</table>
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Add 50ml H2O, Mix, add H2O till 100ml, Filter sterilize and freeze at -20 °C
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Sub2: MC 10x
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<br/>Sub3: Tri-Na Citrate 300mM  
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Ferric NH4 citrate (Sub4) 1ml 0,1ml
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<table border="1">
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Casein Hydrolysate 1g 0,1g
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<tr>
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Potassium Glutamate 2g 0,2g
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<td>Tri-Na Citrate</td>
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Add 50ml H2O, Mix, add H2O till 100ml, Filter sterilize and freeze at -20 °C
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<td>0,8823g</td>
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</tr>
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Sub3: Tri-Na Citrate 300mM  
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<tr>
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Tri-Na Citrate 0,8823g
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<td>H2O</td>
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H2O 10ml
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<td>10ml</td>
 +
</tr>
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</table>
Wrap in aluminum
Wrap in aluminum
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Sub4: Ferric NH4 citrate
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<br/>Sub4: Ferric NH4 citrate
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Ferric NH4 citrate 0,22g
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<table border="1">
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H2O 10ml
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<tr>
 +
<td>Ferric NH4 citrate</td>
 +
<td>0,22g</td>
 +
</tr>
 +
<tr>
 +
<td>H2O</td>
 +
<td>10ml</td>
 +
</tr>
 +
</table>
Wrap in aluminum
Wrap in aluminum

Revision as of 14:44, 17 July 2013

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
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