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User:University of Washington/18 August 2008
From 2008.igem.org
LuxR from AraC and TetR
- miniprepped the ligated plasmid(x4 GFP, x4 LuxR) and submit sequencing.
Yeast Shuttle Vector (Alec)
- Diluted overnight culutres of S. cerevisiae into YEPD to an OD of 0.25
- Grew culture to an OD of 1.0
- Centrifuged cells at 5000 rpm for 5 minutes
- Poured off supernatant, resuspended pellet in 25 mL of sterile dH2O and centrifuged at 5000 rpm for 5 minutes
- Poured off supernatant, resuspended cells in 1 mL of 10x (1 M) LiAc, then transferred to an Eppendorf tube
- Pelleted the cells at 7000 rpm for 15 seconds then removed the supernatant with a pipette
- Resuspended the cells to a final volume of approximately 900 uL with 1x (100 mM) LiAc, then split into 8 Eppendorf tubes
- Vortexed the cell suspension, split the mix into 8 Eppendorf tubes, storing 5 at -80 C
- Pelleted the cells at 7000 rpm for 15 seconds then removed the supernatant with a pipette
- Boiled SS-DNA at ~105 C for 5 minutes then chilled on ice
- Added 240 uL PEG, 36 uL 10x (1 M) LiAc, 40 uL of SS-DNA, 25 uL of LAC amplicon, 12.5 uL of each sample of linearized pAC88
- Resuspended cells, vortexed vigorously, then placed in 30 C for 30 minutes
- Placed tubes in a 42 C water bath for 20 minutes
- Pelleted cells at 4000 rpm for 8 seconds, removed supernatant, resuspended in 400 uL dH2O, and plated on selective media
- Ligated ADH1 vector (J63005) and LacZ insert (J33202)
- Transformed TOP10 cells with J63005-J33202 ligation
- Plated vector/insert ligation on LB+Amp plate and grew overnight at 37 degrees Celsius
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