Team:UNITN-Trento/Protocols

From 2013.igem.org

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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}}
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{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|BioBrick Cloning|<html>
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<span>Prepare the competence medium as follow:</span>
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<table class="tn-sp-table">
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<tr>
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<th>Competence medium (MC completed)</th>
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</tr>
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<tr>
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<td>
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H2O
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</td>
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<TD>
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1.8 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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10x MC
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</td>
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<TD>
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200 ul
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</TD>
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</tr>
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<tr>
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<td>
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MgSO4 1M
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</td>
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<TD>
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6.7 ul
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</TD>
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</tr>
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<tr>
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<td>
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trp 1% (for trp - strains)
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</td>
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<TD>
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10 ul
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</TD>
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</tr>
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</table>
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<table class="tn-sp-table">
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<tr>
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<th>
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MC 10x
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</th>
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</tr>
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<tr>
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<td>
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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>
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14.036 g
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</TD>
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</tr>
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<tr>
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<td>
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KH2PO4
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</td>
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<TD>
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5.239 g
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</TD>
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</tr>
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<tr>
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<td>
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Glucose
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</td>
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<TD>
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20 g
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</TD>
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</tr>
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<tr>
 +
<td>
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Tri-Na Citrate 300 mM
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</td>
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<TD>
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10 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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Ferric NH4 Citrate
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</td>
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<TD>
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1 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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Casein Hydrolysate
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</td>
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<TD>
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1 g
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</TD>
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</tr>
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<tr>
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<td>
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K glutamate
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</td>
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<TD>
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2 g
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</TD>
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</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>
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</table>
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<table class="tn-sp-table">
 +
<tr>
 +
<th>
 +
Tri-Na Citrate 300mM
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</th>
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<td>
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8.823 g
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</td>
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<td>
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in 100 ml H2O
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</td>
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</tr>
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<tr>
 +
<th>
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Ferric NH4 citrate
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</th>
 +
<td>
 +
2.2 g
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</td>
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<td>
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in 100 ml H2O
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</td>
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</tr>
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<tr>
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<td>
 +
--> wrap in aluminium foil!!
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</td>
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</tr>
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</table>
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<br/>
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<ol>
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<li>
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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>
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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>
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<li>
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Grow for additional 2 h at 37 &deg;C;
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</li>
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<li>
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Plate all on selective antibiotic plates, and incubate at 37 &deg;C O/N
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</li>
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</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&deg;C overnight. The day after add 1&micro;L of phosphatase (CIP or SAP) to the vector and incubate for 2 hours at 37&deg;C.
Incubate at 37&deg;C overnight. The day after add 1&micro;L of phosphatase (CIP or SAP) to the vector and incubate for 2 hours at 37&deg;C.
-
<h4>Biobricks Cloning</h4>
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    <h4>Screening</h4>
-
        Incubate at 37&deg;C for 30 minutes. Then disactivate the enzymes at 80&deg;C for 20 minutes.
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-
        <h4>Screening</h4>
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Incubate for 1.5h at 37&deg;C. Run all the digested product on an agarose gel to screen colonies.  
Incubate for 1.5h at 37&deg;C. Run all the digested product on an agarose gel to screen colonies.  
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<br/><br/><hr/><br/>

Revision as of 11:58, 31 July 2013