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Team:Exeter/lab book/proto/4
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<p><b>Digestion Protocol</b></p> | <p><b>Digestion Protocol</b></p> | ||
<br> | <br> | ||
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| - | <table cellpadding="10" cellspacing="10" | + | <table cellpadding="10" cellspacing="10"> |
| + | <tr> | ||
| + | <td colspan="2" align="center"><u>Upstream digest</u></td><td></td><td colspan="2" align="center"><u>Downstream digest</u></td> | ||
<tr> | <tr> | ||
| - | <td>Upstream Part Plasmid</td><td>500 ng</td> | + | <td>Upstream Part Plasmid</td><td>500 ng</td><td></td><td>Upstream Part Plasmid</td><td>500 ng</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>EcoR1-HF</td><td>1 µl</td> | + | <td>EcoR1-HF</td><td>1 µl</td><td></td><td>Xba1</td><td>1 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>Spe1</td><td>1 µl</td> | + | <td>Spe1</td><td>1 µl</td><td></td><td>Pst1</td><td>1 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>10X NEBuffer2</td><td>5 µl</td> | + | <td>10X NEBuffer2</td><td>5 µl</td><td></td><td>10X NEBuffer2</td><td>5 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>100X BSA</td><td>0.5 µl</td> | + | <td>100X BSA</td><td>0.5 µl</td><td></td><td>100X BSA</td><td>0.5 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>H2O</td><td>to 50 µl</td> | + | <td>H2O</td><td>to 50 µl</td><td></td><td>H2O</td><td>to 50 µl</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<p><b>Ligation Protocol</b></p> | <p><b>Ligation Protocol</b></p> | ||
<p>Ligate the Upstream and Downstream Parts into the digested Destination Plasmid.</p> | <p>Ligate the Upstream and Downstream Parts into the digested Destination Plasmid.</p> | ||
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<table cellpadding="10" cellspacing="10"> | <table cellpadding="10" cellspacing="10"> | ||
<tr> | <tr> | ||
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</table> | </table> | ||
| - | + | <table width="980" align="center" cellspacing="20"> | |
| + | <tr align="center"> | ||
| + | <td> | ||
| + | <font color="#57B947" size="1" face="Verdana"> | ||
| + | <p><u>Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley</u> | | ||
| + | <a href="https://igem.org/Team.cgi?id=764" style="color:#57B947" target="_blank"><u>Contact Us</u></a> | | ||
| + | <a href="https://2012.igem.org/Team:Exeter/site_map" style="color:#57B947"><u>Site Map</u></a></p> | ||
| + | </font> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
</body> | </body> | ||
</html> | </html> | ||
Latest revision as of 23:47, 26 September 2012
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Digestion Protocol
Digest the Destination Plasmid with EcoR1-HF and Pst1: The Destination Plasmid DNA should either be prepared with PCR or contain a toxic gene (e.g. ccdb, sacB) in the cloning site to avoid the need for gel purification. The Destination Plasmid should also have a different antibiotic resistance marker from both the plasmid containing the Upstream Part and the plasmid containing the Downstream Part to avoid the need to purify the Upstream and Downstream Parts.
Incubate all three restriction digest reactions at 37 °C for 10 minutes and then heat inactivate at 80 °C for 20 minutes. Ligation Protocol Ligate the Upstream and Downstream Parts into the digested Destination Plasmid.
Incubate at room temperature for 10 minutes and then heat inactivate at 80 °C for 20 minutes. |
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