User:University of Washington/18 August 2008

LuxR from AraC and TetR
- miniprepped the ligated plasmid(x4 GFP, x4 LuxR) and submit sequencing.

LuxR from pLac
-A 6 hr, 25-well assay of the lac promoter in front of GFP, grown in varying concentrations of IPTG and glucose was ran to characterize the behavior of the lac promoter.

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 leucine deficient 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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