Team:Groningen/protocols

From 2013.igem.org

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'''B. subtilis transformation'''
 
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<br/>Losick protocol
 
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<br/>-1 Day: Streak out strain and incubate plate o/n at 37 °C.
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[[:Team:Groningen/protocols/Transformation]]
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<br/>Transformation (D-Day):
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<br/>1. Pick up a nice big colony and drop it in 2 ml of completed MC (1x)(see sub1).
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<br/>2. Grow at 37 °C for 5 hours (more if culture is not really turbid).
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<br/>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)
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<br/>4. Grow for an additional 2 hours at 37 °C.
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<br/>5. Plate all on selective antibiotic plates, and incubates at 37°C o/n.
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<br/>Sub1: Copmpetence medium (MC completed)
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<table border="1">
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<tr>
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<td>H20</td>
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<td>1,8 ml</td>
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<td></td>
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</tr>
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<tr>
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<td>10x MC (sub2)</td>
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<td>200 ul</td>
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<td> filter sterilize</td>
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</tr>
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<tr>
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<td>MgSO4</td>
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<td>6,7 ul</td>
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<td>autoclave sterilize</td>
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</tr>
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<tr>
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<td>Tryptophan 1%</td>
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<td>10 ul</td>
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<td>filter steralize, wrap in Al</td>
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</tr></table>
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<br/>Sub2: MC 10x
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<table border="1">
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<tr>
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<td>for</td>
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<td>100 ml</td>
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<td>10 ml</td>
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</tr>
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<tr>
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<td>K2H PO4</td>
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<td>14,036g</td>
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<td> 1,4036g</td>
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</tr>
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<tr>
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<td>KH2 PO$</td>
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<td>5,239g</td>
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<td>0,5239g</td>
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</tr>
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<tr>
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<td>Glucose</td>
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<td>20g</td>
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<td>2g</td>
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</tr>
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<tr>
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<td>Tri-Na Citrate 300mM(Sub3)</td>
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<td>10ml</td>
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<td>1ml</td>
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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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<br/>Sub3: Tri-Na Citrate 300mM
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<table border="1">
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<tr>
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<td>Tri-Na Citrate</td>
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<td>0,8823g</td>
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</tr>
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<tr>
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<td>H2O</td>
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<td>10ml</td>
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</tr>
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</table>
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Wrap in aluminum
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<br/>Sub4: Ferric NH4 citrate
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<table border="1">
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<tr>
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<td>Ferric NH4 citrate</td>
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<td>0,22g</td>
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</tr>
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<tr>
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<td>H2O</td>
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<td>10ml</td>
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</tr>
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</table>
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Wrap in aluminum
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Revision as of 19:51, 17 July 2013

Team:Groningen/protocols/Transformation

Retrieved from "http://2013.igem.org/Team:Groningen/protocols"