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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.
Team:University_of_Washington/Notebook
