Team:Slovenia/Notebook

From 2012.igem.org

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<h3>Fragment DNA isolation from agarose gel</h3>
<h3>Fragment DNA isolation from agarose gel</h3>
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<p><b>AGAROSE ELECTROPHORESIS</p>
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<p><b>AGAROSE ELECTROPHORESIS</b></p>
<ol>
<ol>
<li>A mixture of different sized DNA fragments was  separated on an agarose gel (from 0.7 to 2% agarose in 1x TAE buffer and 0.1 µg/ml ethidium bromide) at a constant voltage of 100 V. </li>
<li>A mixture of different sized DNA fragments was  separated on an agarose gel (from 0.7 to 2% agarose in 1x TAE buffer and 0.1 µg/ml ethidium bromide) at a constant voltage of 100 V. </li>
<li>UV light (λ = 254 nm) was used to visualize DNA with intercalated ethidium bromide </li>
<li>UV light (λ = 254 nm) was used to visualize DNA with intercalated ethidium bromide </li>
</ol>
</ol>
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</br>
<p><b>FRAGMENT ISOLATION from agarose gel </b></p>
<p><b>FRAGMENT ISOLATION from agarose gel </b></p>
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<ol>
<li>The band with the desired DNA fragment was excised from the gel, using a clean scalpel. </li>
<li>The band with the desired DNA fragment was excised from the gel, using a clean scalpel. </li>
<li>DNA was isolated from the gel slice with GeneJet Gel Extraction Kit according to the manufacturer’s protocol. </li>
<li>DNA was isolated from the gel slice with GeneJet Gel Extraction Kit according to the manufacturer’s protocol. </li>
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<br/>
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<h3>Restriction digest</h3>
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<ol>
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<li>To digest the desired DNA restriction reactions were prepared as follows: </li>
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<span style="margin-left: 30px;><b>for analysis of cloned DNA</b>
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<li>2µl of the appropriate restriction buffer (10X) </li>
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<li>0.5 µL restriction enzyme </li>
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<li>Bring volume to 20 µL with nuclease-free water. </li>
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<li>OR</li>
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<span style="margin-left: 30px;><b>for isolation of specific DNA</b>
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<li>2µl of the appropriate restriction buffer (10X) </li>
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<li>up to 2 µL restriction enzyme </li>
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<li>Bring volume to 50 µL with nuclease-free water. </li></span>
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<li>The sample was incubated at optimal temperature for the restriction enzymes.</li>
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<li>Analysis of fragmented DNA was done by gel electrophoreses. </li>
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<li>Desired DNA fragment was excised and purified using suitable DNA purification kit. </li>
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</ol>
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Revision as of 16:05, 26 September 2012