Team:UNITN-Trento/Protocols
From 2013.igem.org
(Difference between revisions)
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Prepare your reaction and incubate at RT for 2 hours. Transform half of the reaction into 200μL of “homemade” competent cells (DH5α, NEB10β, Novablue or other appropriate strains) following a standard transformation protocol. Plate all the cells. | Prepare your reaction and incubate at RT for 2 hours. Transform half of the reaction into 200μL of “homemade” competent cells (DH5α, NEB10β, Novablue or other appropriate strains) following a standard transformation protocol. Plate all the cells. | ||
</html>|Ligation}} | </html>|Ligation}} | ||
+ | |||
+ | {{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|BioBrick Cloning|<html> | ||
+ | <span>Prepare the competence medium as follow:</span> | ||
+ | <table class="tn-sp-table"> | ||
+ | <tr> | ||
+ | <th>Competence medium (MC completed)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | H2O | ||
+ | </td> | ||
+ | <TD> | ||
+ | 1.8 ml | ||
+ | </TD> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | 10x MC | ||
+ | </td> | ||
+ | <TD> | ||
+ | 200 ul | ||
+ | </TD> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | MgSO4 1M | ||
+ | </td> | ||
+ | <TD> | ||
+ | 6.7 ul | ||
+ | </TD> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | trp 1% (for trp - strains) | ||
+ | </td> | ||
+ | <TD> | ||
+ | 10 ul | ||
+ | </TD> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <table class="tn-sp-table"> | ||
+ | <tr> | ||
+ | <th> | ||
+ | MC 10x | ||
+ | </th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | for 100 ml | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | K2HPO4 3H2O | ||
+ | </td> | ||
+ | <TD> | ||
+ | 14.036 g | ||
+ | </TD> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <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>|biobrick-cloning}} | ||
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Digestion|<html> | {{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Digestion|<html> | ||
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<h4>Classic Cloning - for plasmids</h4> | <h4>Classic Cloning - for plasmids</h4> | ||
Incubate at 37°C overnight. The day after add 1µL of phosphatase (CIP or SAP) to the vector and incubate for 2 hours at 37°C. | Incubate at 37°C overnight. The day after add 1µL of phosphatase (CIP or SAP) to the vector and incubate for 2 hours at 37°C. | ||
- | + | <h4>Screening</h4> | |
- | + | ||
- | + | ||
Incubate for 1.5h at 37°C. Run all the digested product on an agarose gel to screen colonies. | Incubate for 1.5h at 37°C. Run all the digested product on an agarose gel to screen colonies. | ||
<br/><br/><hr/><br/> | <br/><br/><hr/><br/> |
Revision as of 11:58, 31 July 2013