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>
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<!--biobrick cloning protocol -->
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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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<h1>BioBrick cloning</h1>
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<br/>
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<br/>
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<p>
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Prepare the digestion mix as follow:  
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</p>
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<table>
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<tr>
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<td>
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DNA
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</td>
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<td>
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500 ng
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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 NEB Buffer
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</td>
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<td>
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2.5 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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10X BSA
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</td>
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<td>
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2.5 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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E1
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</td>
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<td>
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1 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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E2
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</td>
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<td>
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1 ul
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</td>
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</tr>
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<tr>
<tr>
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<td>
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<th>Competence medium (MC completed)</th>
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H2O
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</tr>
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</td>
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<tr>
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<td>
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<td>
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Up to 25 ul  
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H2O
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</td>
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</td>
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</tr>
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<TD>
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</table>
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1.8 ml
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</TD>
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<p>
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</tr>
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Incubate the reaction mix at 37 &deg;C for 30 min. Disactivate then the enzymes incubating the mix at 80 &deg;C for 20 min.
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<tr>
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The next step will be the ligation of the digestion products. The raction mix is prepared as follow:
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<td>
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</p>
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10x MC
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<table>
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</td>
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<tr>
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<TD>
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<td>
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200 ul
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Insert
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</TD>
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</td>
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</tr>
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<td>
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<tr>
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3 fold excess
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<td>
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</td>
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MgSO4 1M
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</tr>
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</td>
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<tr>
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<TD>
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<td>
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6.7 ul
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Vector
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</TD>
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</td>
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</tr>
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<td>
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<tr>
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40 ng
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<td>
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</td>
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trp 1% (for trp - strains)
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</tr>
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</td>
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<tr>
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<TD>
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<td>
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10 ul
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10X T4 Ligase Buffer
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</TD>
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</td>
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</tr>
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<td>
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</table>
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2 ul
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<table class="tn-sp-table">
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</td>
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<tr>
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</tr>
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<th>
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<tr>
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MC 10x
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<td>
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</th>
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T4 Ligase
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</tr>
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</td>
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<tr>
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<td>
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<td>
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1 ul
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for 100 ml
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</td>
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</td>
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</tr>
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</tr>
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<tr>
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<tr>
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<td>
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<td>
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H2O
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K2HPO4 3H2O
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</td>
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</td>
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<TD>
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<TD>
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Up to 20 ul
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14.036 g
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</TD>
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</TD>
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</tr>
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</tr>
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<tr>
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</table>
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<td>
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<p>
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KH2PO4
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Gently mix the reaction and incubate for 30 min at room temperature. Disactivate the enzymes at 80 &deg;C for 20 min. Transorm 10 ul of the reaction in competent cells.
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</td>
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</p>
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<TD>
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5.239 g
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</TD>
 +
</tr>
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<tr>
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<td>
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Glucose
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</td>
 +
<TD>
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20 g
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</TD>
 +
</tr>
 +
<tr>
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<td>
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Tri-Na Citrate 300 mM
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</td>
 +
<TD>
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10 ml
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</TD>
 +
</tr>
 +
<tr>
 +
<td>
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Ferric NH4 Citrate
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</td>
 +
<TD>
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1 ml
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</TD>
 +
</tr>
 +
<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>
 +
</tr>
 +
<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>
 +
</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 class="tn-sp-table">
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<tr>
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<th>
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Tri-Na Citrate 300mM
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</th>
 +
<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>
 +
</tr>
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<tr>
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<th>
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Ferric NH4 citrate
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</th>
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<td>
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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>
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--> 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>|subtilis-transformation}}
</html>|subtilis-transformation}}
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Prepare the digestion mix as follow:  
Prepare the digestion mix as follow:  
</p>
</p>
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<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>
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<table>
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<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


Retrieved from "http://2013.igem.org/Team:UNITN-Trento/Protocols"