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User:University of Washington/22 July 2008
From 2008.igem.org
RP4 Conjugation
Test conjugation protocol #2
·DH5alpha RP4 + pCS26-pac
·DH5alpha + ""
Glycerol Stock DH5alpha RP4
LuxR from AraC and TetR
- Made 6 overnight cultures for AraC plasmid (R0080).
- QuikChange Mutagenesis
| Materials | Reaction#1 | Reaction#2 | negative | positive |
|---|---|---|---|---|
| AraC plasmid | 39.4 ul | 39.5 ul | 39.4 ul | 5 ul(only had that much left) |
| 10X pfuTurbo reaction buffer | 5 ul | 5 ul | 5 ul | 5 ul |
| dNTP mix | 1 ul | 1 ul | 1 ul | 1 ul |
| primer AraC-F | 0.806 ul 1F | 0.728 ul 2F | - | 0.806 ul 1F |
| primer AraC-R | 0.806 ul 1R | 0.728 ul 2R | - | 0.806 ul 1R |
| DMSO | 3 ul | 3 ul | 3 ul | 3 ul |
| ddH2O | - | - | 1.6 ul | 34.4 ul |
- add 1 ul pfuTurbo in each tubes
- Temperature Cycle(There are small changes from our protocol page).
| Segment | Cycles | Temperature | Time |
|---|---|---|---|
| 1 | 1 | 95°C | 2 minute |
| 2 | 18 | 95°C | 50 seconds |
| 60°C | 50 seconds | ||
| 68°C | 3 mins | ||
| 3 | 1 | 68°C | 7 minutes |
| let it sit in 4°C |
- added Dpn1 in each tube EXCEPT positive control, let the reaction sit in 37 degree Celsius.
- After 2.5 hours, the DNA product was taken out to do PRC purification. (30 EB Buffer at the end)
- Electroporated the purified DNA into XL1-Blue. 45 ul cells + 2 ul DNA (4 tubes)
- Let it sit 30 mins and then inoculated onto LB Agar + Amp plate. Stored in 37 degree Celsius incubator.
Lambda Red Recombineering of RP4
Troubleshooting of PCR continued. Performed 2 sets of reactions in parallel -- one using the iGEM lab's reagents and the other using grad advisor's reagents. My colony PCR failed, while Ingrid's was successful. Both of our control reactions on confirmed part J04430 were successful, though Ingrid got better yield. Also got amplification of C0161. Process of elimination leads me to believe that magnesium chloride concentration is not optimized.
Team:University_of_Washington/Notebook
