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Team:Berkeley Wetlab/Passenger: MGFP
From 2009.igem.org
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(→Day 4: Run BCA protein quantification assay) |
(→MGFP-5 BCA Protein Quantification Assay) |
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'' Experiment run with 5x replicate of each condition'' | '' Experiment run with 5x replicate of each condition'' | ||
| - | === Day 1: Grow | + | === Day 1: Grow Cells === |
* Pick cells, using pin tool, with appropriate constructs from frozen stock plates into 96-well blocks with 1 ml of LB+AC media | * Pick cells, using pin tool, with appropriate constructs from frozen stock plates into 96-well blocks with 1 ml of LB+AC media | ||
* Place blocks in 37C shaker overnight to grow to saturation | * Place blocks in 37C shaker overnight to grow to saturation | ||
| - | === Day 2: | + | === Day 2: Induce Cells === |
* Set up 96-well blocks with 1 ml of LB+AC+arabinose media: | * Set up 96-well blocks with 1 ml of LB+AC+arabinose media: | ||
** Add 1 ul of arabinose at a concentration of (100mg/ml) to one ml of LB+AC media (1: 1000) | ** Add 1 ul of arabinose at a concentration of (100mg/ml) to one ml of LB+AC media (1: 1000) | ||
* Take plates with constructs out of the 37C incubator and add 10 ul of each construct to the LB+AC+arabinose media to induce | * Take plates with constructs out of the 37C incubator and add 10 ul of each construct to the LB+AC+arabinose media to induce | ||
| - | * | + | * Incubate blocks at 37C in shaker overnight. |
| - | + | ||
=== Day 3: Incubate in Artificial Seawater === | === Day 3: Incubate in Artificial Seawater === | ||
| - | + | * Remove blocks from incubator | |
| - | + | * Pellet cells at 5400 rpm for 7 minutes | |
| + | * Remove supernatant by flicking block over a sink (swiftly inverting block in one motion) | ||
| + | * Re-suspend cells in 100 ul of artificial seawater | ||
| + | * Transfer 50 ul of cell suspensions with multi-pipette to labeled flat bottom 96-well costar plates (polystyrene plates) | ||
| + | * Cover plates with plastic film and let cells settle overnight | ||
| + | |||
=== Day 4: Run BCA Protein Quantification Assay === | === Day 4: Run BCA Protein Quantification Assay === | ||
Revision as of 18:24, 18 October 2009
Contents |
MGFP-5 BCA Protein Quantification Assay
This assay tests for the presence of mgfp-5 displayed on the surface of E. coli through its ability to bind to the bottom of polystyrene plates.
Constructs used:
- mgfp-5 + spacers + displayers (79)
- mgfp-5 + displayers (14)
- displayers only (15)
Experiment run with 5x replicate of each condition
Day 1: Grow Cells
- Pick cells, using pin tool, with appropriate constructs from frozen stock plates into 96-well blocks with 1 ml of LB+AC media
- Place blocks in 37C shaker overnight to grow to saturation
Day 2: Induce Cells
- Set up 96-well blocks with 1 ml of LB+AC+arabinose media:
- Add 1 ul of arabinose at a concentration of (100mg/ml) to one ml of LB+AC media (1: 1000)
- Take plates with constructs out of the 37C incubator and add 10 ul of each construct to the LB+AC+arabinose media to induce
- Incubate blocks at 37C in shaker overnight.
Day 3: Incubate in Artificial Seawater
- Remove blocks from incubator
- Pellet cells at 5400 rpm for 7 minutes
- Remove supernatant by flicking block over a sink (swiftly inverting block in one motion)
- Re-suspend cells in 100 ul of artificial seawater
- Transfer 50 ul of cell suspensions with multi-pipette to labeled flat bottom 96-well costar plates (polystyrene plates)
- Cover plates with plastic film and let cells settle overnight
