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EPF-Lausanne/30 July 2008
From 2008.igem.org
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<p>We started a 2 step PCR too get LuxR, lasR, RhlR genes. We intend to convert them to proteins invitro to test their binding affinity to their DNA sites. | <p>We started a 2 step PCR too get LuxR, lasR, RhlR genes. We intend to convert them to proteins invitro to test their binding affinity to their DNA sites. | ||
1st step worked fine. 2nd stop should finish this evening. We have to order the "ribosomal solution" to continue. | 1st step worked fine. 2nd stop should finish this evening. We have to order the "ribosomal solution" to continue. | ||
| - | We | + | We used more DNA and primers because 1st try didn't work. Here is our protocal. Normaly 1ul of DNA and primers. |
</p> | </p> | ||
| + | <h3>Protocal</h3> | ||
| + | <table width="207" border="1"> | ||
| + | <tr> | ||
| + | <td width="128">dNTP</td> | ||
| + | <td width="63"><div align="center">1ul</div></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>10x Buffer + mgcl2</td> | ||
| + | <td><div align="center">5ul</div></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>DNApoly</td> | ||
| + | <td><div align="center">1ul</div></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>3'primer</td> | ||
| + | <td><div align="center">5ul</div></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>5'primer</td> | ||
| + | <td><div align="center">5ul</div></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>template</td> | ||
| + | <td><div align="center">5ul</div></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>H2O</td> | ||
| + | <td><div align="center">28ul</div></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>---------------------</td> | ||
| + | <td><div align="center">----------</div></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Total</td> | ||
| + | <td><div align="center">50</div></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
<p> | <p> | ||
We our finishing the exaxt design of our plasmids. Extracting LacI from the Ron-Weiss plasmid and making it a part will probably need new primers(so we can add the restriction sites). | We our finishing the exaxt design of our plasmids. Extracting LacI from the Ron-Weiss plasmid and making it a part will probably need new primers(so we can add the restriction sites). | ||
</p> | </p> | ||
Same has to be done for incorporating RhlI in the GFP operon on the pHD plasmids. We may use PciI to cut the plasmid in one location: 3382. But we have to check if we were the terminator is. May have to digest out the GFP and reinstert a GFP+RhlI operon. | Same has to be done for incorporating RhlI in the GFP operon on the pHD plasmids. We may use PciI to cut the plasmid in one location: 3382. But we have to check if we were the terminator is. May have to digest out the GFP and reinstert a GFP+RhlI operon. | ||
Revision as of 13:49, 30 July 2008
We started a 2 step PCR too get LuxR, lasR, RhlR genes. We intend to convert them to proteins invitro to test their binding affinity to their DNA sites. 1st step worked fine. 2nd stop should finish this evening. We have to order the "ribosomal solution" to continue. We used more DNA and primers because 1st try didn't work. Here is our protocal. Normaly 1ul of DNA and primers.
Protocal
| dNTP | 1ul |
| 10x Buffer + mgcl2 | 5ul |
| DNApoly | 1ul |
| 3'primer | 5ul |
| 5'primer | 5ul |
| template | 5ul |
| H2O | 28ul |
| --------------------- | ---------- |
| Total | 50 |
We our finishing the exaxt design of our plasmids. Extracting LacI from the Ron-Weiss plasmid and making it a part will probably need new primers(so we can add the restriction sites).
Same has to be done for incorporating RhlI in the GFP operon on the pHD plasmids. We may use PciI to cut the plasmid in one location: 3382. But we have to check if we were the terminator is. May have to digest out the GFP and reinstert a GFP+RhlI operon.
