From 2014.igem.org
(Difference between revisions)
|
|
| Line 1: |
Line 1: |
| - | {{:Team:Goettingen/header}}
| |
| - | {{:Team:Goettingen/proLP}}
| |
| | | | |
| - | <html>
| |
| - | <div class="proRP">
| |
| - | <h3 id="rest_DNA">Restriction of DNA</h3>
| |
| - | <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 />
| |
| - | 2. Incubate for at least 1 hour at 37°C (or enzyme specific temperature).<br />
| |
| - | 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 />
| |
| - | 4. Stop reaction by heat-inactivation at 65°C for 5 minutes.<br /><br /></p>
| |
| - |
| |
| - | <h3 id="liga_DNA">Ligation of DNA fragments</h3>
| |
| - | <p>1. Measure the concentration of fragments which should be used for the ligation reaction.<br />
| |
| - | 2. Calculate the ratio between insert and backbone (1:1) using the following formula:<br />
| |
| - | 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 />
| |
| - | 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>
| |
| - |
| |
| - | <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>
| |
| - | </div>
| |
| - | <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>
| |
| - |
| |
| - | </html>
| |
Latest revision as of 12:33, 17 October 2014
"
