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> |
- | + | <table class="tn-sp-table"> | |
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<tr> | <tr> | ||
- | < | + | <th>Competence medium (MC completed)</th> |
- | H2O | + | </tr> |
- | + | <tr> | |
- | <td> | + | <td> |
- | + | H2O | |
- | </td> | + | </td> |
- | + | <TD> | |
- | </ | + | 1.8 ml |
- | + | </TD> | |
- | < | + | </tr> |
- | + | <tr> | |
- | + | <td> | |
- | </ | + | 10x MC |
- | <table> | + | </td> |
- | <tr> | + | <TD> |
- | <td> | + | 200 ul |
- | + | </TD> | |
- | </ | + | </tr> |
- | <td> | + | <tr> |
- | + | <td> | |
- | </td> | + | MgSO4 1M |
- | + | </td> | |
- | + | <TD> | |
- | <td> | + | 6.7 ul |
- | + | </TD> | |
- | </ | + | </tr> |
- | <td> | + | <tr> |
- | + | <td> | |
- | </td> | + | trp 1% (for trp - strains) |
- | + | </td> | |
- | + | <TD> | |
- | <td> | + | 10 ul |
- | + | </TD> | |
- | </ | + | </tr> |
- | <td> | + | </table> |
- | 2 | + | <table class="tn-sp-table"> |
- | </td> | + | <tr> |
- | + | <th> | |
- | < | + | MC 10x |
- | < | + | </th> |
- | + | </tr> | |
- | + | <tr> | |
- | + | <td> | |
- | + | for 100 ml | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr> | |
- | + | <td> | |
- | H2O | + | K2HPO4 3H2O |
- | + | </td> | |
- | < | + | <TD> |
- | + | 14.036 g | |
- | + | </TD> | |
- | + | </tr> | |
- | + | <tr> | |
- | </table> | + | <td> |
- | < | + | KH2PO4 |
- | + | </td> | |
- | </ | + | <TD> |
+ | 5.239 g | ||
+ | </TD> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | Glucose | ||
+ | </td> | ||
+ | <TD> | ||
+ | 20 g | ||
+ | </TD> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | Tri-Na Citrate 300 mM | ||
+ | </td> | ||
+ | <TD> | ||
+ | 10 ml | ||
+ | </TD> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | Ferric NH4 Citrate | ||
+ | </td> | ||
+ | <TD> | ||
+ | 1 ml | ||
+ | </TD> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | Casein Hydrolysate | ||
+ | </td> | ||
+ | <TD> | ||
+ | 1 g | ||
+ | </TD> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | K glutamate | ||
+ | </td> | ||
+ | <TD> | ||
+ | 2 g | ||
+ | </TD> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Mix everything in 40-50 ml H2O, then adjust to 100 ml, filter sterilize, freeze at -20 C</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table class="tn-sp-table"> | ||
+ | <tr> | ||
+ | <th> | ||
+ | Tri-Na Citrate 300mM | ||
+ | </th> | ||
+ | <td> | ||
+ | 8.823 g | ||
+ | </td> | ||
+ | <td> | ||
+ | in 100 ml H2O | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th> | ||
+ | Ferric NH4 citrate | ||
+ | </th> | ||
+ | <td> | ||
+ | 2.2 g | ||
+ | </td> | ||
+ | <td> | ||
+ | in 100 ml H2O | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | --> wrap in aluminium foil!! | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br/> | ||
+ | <ol> | ||
+ | <li> | ||
+ | Pick up a nice big colony and drop it in 2 ml of completed MC (1x) (see below); | ||
+ | </li> | ||
+ | <li> | ||
+ | Grow at 37 °C for 5 hours (or more if culture is not really turbid); | ||
+ | </li> | ||
+ | <li> | ||
+ | Mix 400 ul of culture with DNA (usually 1 ug) in fresh tube (i.e. 15 ml tubes losely closed); | ||
+ | </li> | ||
+ | <li> | ||
+ | Grow for additional 2 h at 37 °C; | ||
+ | </li> | ||
+ | <li> | ||
+ | Plate all on selective antibiotic plates, and incubate at 37 °C O/N | ||
+ | </li> | ||
+ | </ol> | ||
</html>|subtilis-transformation}} | </html>|subtilis-transformation}} | ||
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Prepare the digestion mix as follow: | Prepare the digestion mix as follow: | ||
</p> | </p> | ||
- | <table> | + | <table class="tn-sp-table"> |
<tr> | <tr> | ||
<td> | <td> | ||
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The next step will be the ligation of the digestion products. The raction mix is prepared as follow: | The next step will be the ligation of the digestion products. The raction mix is prepared as follow: | ||
</p> | </p> | ||
- | <table> | + | <table class="tn-sp-table"> |
<tr> | <tr> | ||
<td> | <td> | ||
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</TD> | </TD> | ||
</tr> | </tr> | ||
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</table> | </table> | ||
<p> | <p> |
Revision as of 13:02, 31 July 2013