Team:Macquarie Australia/Protocols/ligations

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<html>
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<center><h1>Ligation Procedure</h1></center>
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<p>A reaction mixture was prepared containing,<br>
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<center><table border="3" cellpadding="4" cellspacing="0">
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<tr><td>Element</td>
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<td>Volume</td></tr>
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<tr><td>Upstream Digestion part</td>
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<td>2 &micro;L</td></tr>
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<tr><td>Downstream Digestion part</td>
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<td>2 &micro;L</td></tr>
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<tr><td>Destination Plasmid</td>
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<td>1 &micro;L</td></tr>
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<tr><td>10X T4 DNA ligase buffer</td>
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<td>2 &micro;L</tr></td>
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<tr><td>T4 DNA ligase</td>
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<td>1 &micro;L</td></tr>
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<tr><td>H<sub>2</sub>O</td>
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<td>12 &micro;L</td></tr>
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</table></center>
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</p>
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<p>We incubated each ligation mix at 30&deg;C for 30 minutes, followed by heat inactivation at 80&deg;C for 20 minutes.</p>
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<p>4 &micro;L  of the ligation product was transformed into 100 &micro;L  of competent <i>E. coli</i>, the transformants were then incubated for one hour.</p>
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</html>

Latest revision as of 13:08, 25 September 2012



Ligation Procedure

A reaction mixture was prepared containing,

Element Volume
Upstream Digestion part 2 µL
Downstream Digestion part 2 µL
Destination Plasmid 1 µL
10X T4 DNA ligase buffer 2 µL
T4 DNA ligase 1 µL
H2O 12 µL

We incubated each ligation mix at 30°C for 30 minutes, followed by heat inactivation at 80°C for 20 minutes.

4 µL of the ligation product was transformed into 100 µL of competent E. coli, the transformants were then incubated for one hour.

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