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Team:Cambridge/Protocols
From 2009.igem.org
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(Cells should be at a final volume of ~3 x 10^10 cells.ml^-1) | (Cells should be at a final volume of ~3 x 10^10 cells.ml^-1) | ||
| + | |||
| + | '''Competant cells Transformation''' | ||
| + | |||
| + | *Electrocompetent cells thawed on ice | ||
| + | *Prepare vector DNA on ice | ||
| + | *Biobricks | ||
| + | **With pipette tip, punch hole through foil cover into designated well | ||
| + | **Add 20uL DIW | ||
| + | **We will be removing about 2L; the rest needs to go in an eppendorf, labeled with biobrick number, and stored at -20 degrees C | ||
| + | *Violacein and melanin need to be thawed | ||
| + | *Vector DNA pipetted into chilled 2mm separation electrocuvette = 4 total | ||
| + | *2uL of biobricks | ||
| + | *? uL of melanin and violacein plasmid | ||
| + | *Add 45 uL Competent cells (Duncan’s suggestion was 0.5L DNA to 40 L cells) | ||
| + | *Tap electrocuvette gently to evenly spread mixture in the electrocuvette gap with no air bubbles | ||
| + | *Thoroughly dry the cuvette | ||
| + | *1.68 kV (need to ask James exactly what voltage) passed across cuvette, 5.1-5.4 time constant at 200 ohms and 25 F | ||
| + | *Add 0.25 mL SOC liquid medium (Duncan suggests 100L) to electrocuvette | ||
| + | *Transfer culture to chilled eppendorf, WAIT 2 MINUTES | ||
| + | *Incubate at 37 degrees C for 60 minutes | ||
| + | *Pipette 200L onto a selective LB agar plate, spread with blue spreader, do duplicates, 4 separate inoculums | ||
| + | *orange genes biobrick: ampicillin | ||
| + | *promoter for orange genes biobrick: ampicillin | ||
| + | *melanin: ampicillin, copper, and tyrosine | ||
| + | *violacin: trimethoprim NOT MADE YET | ||
| + | *Do 1:10 dilution with SDW into a new eppendorf | ||
| + | *Pippete 200L onto a selective LB agar plate, spread with blue spreader do duplicates, 4 separate inoculums | ||
Revision as of 08:53, 23 July 2009
Producing competent cells
Starting from a single colony on a plate:
- Transfer colony into 50ml liquid LB media and leave in a 200rpm shaking incubator overnight
- Take 10ml of the cultture and innoculate into larger LB volume and grow in shaking incubator until OD600 of 1.5 (4 hours?)
- Put culture on ice for 30 minutes
- Centrifuge at 4000g for 6 minutes
- Remove supernatent and resuspend cells in an equal volume of ice-cold 0.1mM HEPES
- Repeat centrifugation
- Resuspend cells in 0.5 volume ice-cold 0.1mM HEPES
- Repeat centrifugation
- Resuspend cells in ice-cold 10% glycerol (20ml)
- Combine to form two tubes of 40ml glycerol
- Repeat centrifugation
- Resuspend in ice-cold glycerol (3ml)
- Divide cells into 100ul aliquots and store at -80
(Cells should be at a final volume of ~3 x 10^10 cells.ml^-1)
Competant cells Transformation
- Electrocompetent cells thawed on ice
- Prepare vector DNA on ice
- Biobricks
- With pipette tip, punch hole through foil cover into designated well
- Add 20uL DIW
- We will be removing about 2L; the rest needs to go in an eppendorf, labeled with biobrick number, and stored at -20 degrees C
- Violacein and melanin need to be thawed
- Vector DNA pipetted into chilled 2mm separation electrocuvette = 4 total
- 2uL of biobricks
- ? uL of melanin and violacein plasmid
- Add 45 uL Competent cells (Duncan’s suggestion was 0.5L DNA to 40 L cells)
- Tap electrocuvette gently to evenly spread mixture in the electrocuvette gap with no air bubbles
- Thoroughly dry the cuvette
- 1.68 kV (need to ask James exactly what voltage) passed across cuvette, 5.1-5.4 time constant at 200 ohms and 25 F
- Add 0.25 mL SOC liquid medium (Duncan suggests 100L) to electrocuvette
- Transfer culture to chilled eppendorf, WAIT 2 MINUTES
- Incubate at 37 degrees C for 60 minutes
- Pipette 200L onto a selective LB agar plate, spread with blue spreader, do duplicates, 4 separate inoculums
- orange genes biobrick: ampicillin
- promoter for orange genes biobrick: ampicillin
- melanin: ampicillin, copper, and tyrosine
- violacin: trimethoprim NOT MADE YET
- Do 1:10 dilution with SDW into a new eppendorf
- Pippete 200L onto a selective LB agar plate, spread with blue spreader do duplicates, 4 separate inoculums
