Team:UNITN-Trento/Protocols

From 2013.igem.org

(Difference between revisions)
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{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|B. subtilis transformation (from Groeningen)|<html>
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|B. subtilis transformation (from Groeningen)|<html>
 +
<span>Prepare the competence medium as follow:</span>
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<table>
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<tr>
 +
<th>Competence medium (MC completed)</th>
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</tr>
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<tr>
 +
<td>
 +
H2O
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</td>
 +
<TD>
 +
1.8 ml
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</TD>
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</tr>
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<tr>
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<td>
 +
10x MC
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</td>
 +
<TD>
 +
200 ul
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</TD>
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</tr>
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<tr>
 +
<td>
 +
MgSO4 1M
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</td>
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<TD>
 +
6.7 ul
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</TD>
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</tr>
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<tr>
 +
<td>
 +
trp 1% (for trp - strains)
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</td>
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<TD>
 +
10 ul
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</TD>
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</tr>
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</table>
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<table>
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<tr>
 +
<th>
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MC 10x
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</th>
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</tr>
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<tr>
 +
<td>
 +
for 100 ml
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</td>
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</tr>
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<tr>
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<td>
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K2HPO4 3H2O
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</td>
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<TD>
 +
14.036 g
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</TD>
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</tr>
 +
<tr>
 +
<td>
 +
KH2PO4
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</td>
 +
<TD>
 +
5.239 g
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</TD>
 +
</tr>
 +
<tr>
 +
<td>
 +
Glucose
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</td>
 +
<TD>
 +
20 g
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</TD>
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</tr>
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<tr>
 +
<td>
 +
Tri-Na Citrate 300 mM
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</td>
 +
<TD>
 +
10 ml
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</TD>
 +
</tr>
 +
<tr>
 +
<td>
 +
Ferric NH4 Citrate
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</td>
 +
<TD>
 +
1 ml
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</TD>
 +
</tr>
 +
<tr>
 +
<td>
 +
Casein Hydrolysate
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</td>
 +
<TD>
 +
1 g
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</TD>
 +
</tr>
 +
<tr>
 +
<td>
 +
K glutamate
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</td>
 +
<TD>
 +
2 g
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</TD>
 +
</tr>
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<tr>
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<td>Mix everything in 40-50 ml H2O, then adjust to 100 ml, filter sterilize, freeze at -20 C</td>
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</tr>
 +
</table>
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<table>
 +
<tr>
 +
<th>
 +
Tri-Na Citrate 300mM
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</th>
 +
<td>
 +
8.823 g
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</td>
 +
<td>
 +
in 100 ml H2O
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</td>
 +
</tr>
 +
<tr>
 +
<th>
 +
Ferric NH4 citrate
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</th>
 +
<td>
 +
2.2 g
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</td>
 +
<td>
 +
in 100 ml H2O
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</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
--> wrap in aluminium foil!!
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</td>
 +
</tr>
 +
</table>
 +
<br/>
 +
<ol>
 +
<li>
 +
Pick up a nice big colony and drop it in 2 ml of completed MC (1x) (see below);
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</li>
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<li>
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Grow at 37 &deg;C for 5 hours (or more if culture is not really turbid);
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</li>
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<li>
 +
Mix 400 ul of culture with DNA (usually 1 ug) in fresh tube (i.e. 15 ml tubes losely closed);
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</li>
 +
<li>
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Grow for additional 2 h at 37 &deg;C;
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</li>
 +
<li>
 +
Plate all on selective antibiotic plates, and incubate at 37 &deg;C O/N
 +
</li>
 +
</ol>
</html>|subtilis-transformation}}
</html>|subtilis-transformation}}

Revision as of 11:37, 31 July 2013


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