Team:Goettingen/protocol DNA

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4. Incubate your reaction mix for 1 hour at room temperature or over night at 16°C.<br />
4. Incubate your reaction mix for 1 hour at room temperature or over night at 16°C.<br />
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>
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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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<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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Revision as of 11:57, 16 October 2014

Restriction of DNA

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.
2. Incubate for at least 1 hour at 37°C (or enzyme specific temperature).
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.
4. Stop reaction by heat-inactivation at 65°C for 5 minutes.

Ligation of DNA fragments

1. Measure the concentration of fragments which should be used for the ligation reaction.
2. Calculate the ratio between insert and backbone (1:1) using the following formula:
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.
4. Incubate your reaction mix for 1 hour at room temperature or over night at 16°C.
5. Use at least 4 µl of the reaction mixture for transformation. (Previous purification of the ligation product can avoid false positive colonies!)

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