"
EPF-Lausanne/30 July 2008
From 2008.igem.org
Contents |
2 Step PCR Protocol
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 except for LasR, but we were not sure the gene was actually in the plasmid. 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.
1st Step
| dNTP | 1ul |
| 10x Buffer + mgcl2 | 5ul |
| DNApoly | 1ul |
| 3'primer | 5ul |
| 5'primer | 5ul |
| template | 5ul |
| H2O | 28ul |
| --------------------- | ---------- |
| Total | 50 |
2nd Step
Dilute primers to 250nM each. The template is the previous PCR mix
| dNTP | 1ul |
| 10x Buffer + mgcl2 | 5ul |
| DNApoly | 1ul |
| Primer mix | 1ul |
| template | 5ul |
| H2O | 41.5ul |
| --------------------- | ---------- |
| Total | 50 |
Launch for 10 cycles and then add the final primers. 1ul of each and launch for an other 30 cycles.
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.
Digestion for biobrick assembly :
We started at both ends so that if there is a problem, we still advance somehow.
- LuxI(F1610) and RFP(E1010)
- constitutive promoter(I14033) and RhlR(I1466)
We assemble some small bricks together to gain time
- ptetR(R0040) and RBS(B0034)
- PRhlR(R0071) and RBS(B0034)
