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User:University of Washington/18 August 2008
From 2008.igem.org
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- miniprepped the ligated plasmid(x4 GFP, x4 LuxR) and submit sequencing. | - 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 | ||
---- | ---- | ||
Back to [[Team:University_of_Washington/Notebook#Notebook]] | Back to [[Team:University_of_Washington/Notebook#Notebook]] | ||
Revision as of 03:48, 19 August 2008
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
Back to Team:University_of_Washington/Notebook#Notebook
