Team:Goettingen/protocol DNA

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<div class="proRP">
 
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      <h2 id="rest_DNA">Restriction of DNA</h2>
 
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      <p>1. Use up to 1 µg of DNA in a 20 µl reaction volume. Add 1 µl enzyme, 2 µl 10x recommended buffer and add up to 20 µl water.<br />
 
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2. Incubate for at least 1 hour at 37°C (or enzyme specific temperature).<br />
 
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3. Following the incubation, add 1.5 µl SAP (shrimp alkaline phosphatase) to plasmid backbones for 5’‑dephosphorylation and incubate again at 37°C for 10-30 minutes.<br />
 
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4. Stop reaction by heat-inactivation at 65°C for 5 minutes.<br /><br /></p>
 
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      <h2 id="liga_DNA">Ligation of DNA fragments</h2>
 
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      <p>1. Measure the concentration of fragments which should be used for the ligation reaction.<br />
 
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2. Calculate the ratio between insert and backbone (1:1) using the following formula:<br />
 
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3. Pipette all things together. Use 2 µl of 10x T4 Ligation buffer (stored at -20°C, ATP containing) and at least 1 µl T4 ligase.<br />
 
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4. Incubate your reaction mix for 1 hour at room temperature or over night at 16°C.<br />
 
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5. Use at least 4 µl of the reaction mixture for transformation. (Previous purification of the ligation product can avoid false positive colonies!)<br /><br /></p>
 
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        <h2 id="seam_clone">SEAMLESS Cloning</h2>
 
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        <p>
 
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1. Construct primers for the desired construct with 15 bp overhangs for homologous recombination later.  <br />
 
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2. Amplify DNA fragments, purify them and measure concentration.<br />
 
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3. Calculate the amount of PCR product amount you need with the formula:<br />
 
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4. Pipette 100 ng of vector and the calculated amount of insert together. Scale down the reaction from 20 µl to the smallest volume possible. <br />
 
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5. Add 5 fold buffer and last the 10 fold enzyme mix. <br />
 
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6. Incubate for 30 min at room temperature.<br />
 
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7. Cool the sample on ice for not more than 5 minutes.<br />
 
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8. Use the whole sample for transformation.<br /><br />
 
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</p>
 
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</div>
 
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<div id="footerbanner"><a href="https://2014.igem.org/Team:Goettingen/team_sponsors"><img src="https://static.igem.org/mediawiki/2014/7/7a/Goettingen_footer_banner.png" /></a></div>
 
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</html>
 

Latest revision as of 12:33, 17 October 2014