User:University of Washington/18 August 2008

(Difference between revisions)

Revision as of 03:54, 19 August 2008

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

- 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

@@ Line 31: / Line 31: @@
 - 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
+- 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)