Team:Macquarie Australia/Protocols/Ligation

From 2013.igem.org

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<h1>Ligation Procedure</h1>
<h1>Ligation Procedure</h1>
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.datagrid {border-collapse: collapse; text-align: left; width: 100%;} .datagrid {font: normal 12px/150% Verdana, Arial, Helvetica, sans-serif; background: #fff; overflow: hidden; }.datagrid table td, .datagrid table th { padding: 3px 12px; }.datagrid table thead th {background:-webkit-gradient( linear, left top, left bottom, color-stop(0.05, #991821), color-stop(1, #80141C) );background:-moz-linear-gradient( center top, #991821 5%, #80141C 100% );filter:progid:DXImageTransform.Microsoft.gradient(startColorstr='#991821', endColorstr='#80141C');background-color:#991821; color:#FFFFFF; font-size: 14px; font-weight: bold; border-left: 1px solid #B01C26; } .datagrid table thead th:first-child { border: none; }.datagrid table tbody td { color: #000000; border-left: 1px solid #DBDBDB;font-size: 12px;font-weight: normal; }.datagrid table tbody .alt td { background: #303030; color: #FFFFFF; }.datagrid table tbody td:first-child { border-left: none; }.datagrid table tbody tr:last-child td { border-bottom: none; }
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A reaction mixture was prepared containing,
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<b>Prepare</b> the following reaction mixture
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Element Volume
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Upstream Digestion part 2 µL
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Downstream Digestion part 2 µL
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Destination Plasmid 1 µL
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10X T4 DNA ligase buffer 2 µL
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T4 DNA ligase 1 µL
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H2O 12 µL
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<thead><tr><th>Element </th><th>Volume</th></tr></thead>
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<tbody><tr><td>Upstream Digestion part </td><td>2 µL</td></tr>
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<tr class="alt"><td>Downstream Digestion part</td><td>2 µL</td></tr>
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<tbody><tr><td>Destination Plasmid</td><td>1 µL</td></tr>
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<tr class="alt"><td>10X T4 DNA ligase buffer</td><td>2 µL</td></tr>
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<tbody><tr><td>T4 DNA ligase</td><td>1 µL</td></tr>
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<tr class="alt"><td>H2O</td><td>12 µL</td></tr>
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Incubate each ligation mix at 30°C for 30 minutes, followed by heat inactivation at 80°C for 20 minutes.
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We incubated each ligation mix at 30°C for 30 minutes, followed by heat inactivation at 80°C for 20 minutes.
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transform 4 µL of the ligation product into 100 µL of competent E. coli, then incubate the transformants for one hour.
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<br><br>
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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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[[File:LigationQLD.jpg|500px|thumb|center|Ligation]]

Latest revision as of 07:18, 13 September 2013


Ligation Procedure


Prepare the following reaction mixture

Element Volume
Upstream Digestion part 2 µL
Downstream Digestion part2 µL
Destination Plasmid1 µL
10X T4 DNA ligase buffer2 µL
T4 DNA ligase1 µL
H2O12 µL

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

transform 4 µL of the ligation product into 100 µL of competent E. coli, then incubate the transformants for one hour.


Ligation
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