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| - | {{:Team:Goettingen/header}}
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| - | {{:Team:Goettingen/proLP}}
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| - | <div class="proRP">
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| - | <h3 id="rest_DNA">Restriction of DNA</h3>
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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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| - | <h3 id="liga_DNA">Ligation of DNA fragments</h3>
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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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| - | <h3 id="seam_clone">SEAMLESS Cloning</h3>
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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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| - | <b>ng of insert = (2 x bp of insert x 100 (ng vector)) / bp of vector</b><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><a href="https://2014.igem.org/Team:Goettingen/protocol_PCR" class="button_pre"><b>Previous</b></a><a href="https://2014.igem.org/Team:Goettingen/protocol_Plasmid_Con" class="button_next"><b>Next</b></a></div>
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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>
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