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Team:Cambridge/Protocols
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{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--> | {{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--> | ||
| - | + | = Protocols = | |
| - | + | == Producing competent cells == | |
Starting from a single colony on a plate: | Starting from a single colony on a plate: | ||
| Line 21: | Line 21: | ||
(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 | *Electrocompetent cells thawed on ice | ||
| Line 40: | Line 40: | ||
*Incubate electrocuvettes at 37 degrees C for 60 minutes | *Incubate electrocuvettes at 37 degrees C for 60 minutes | ||
*Pipette 150uL onto a (warmed) selective LB agar plate, spread with blue spreader | *Pipette 150uL onto a (warmed) selective LB agar plate, spread with blue spreader | ||
| - | * | + | *Orange genes biobrick: ampicillin |
| - | * | + | *Promoter for orange genes biobrick: ampicillin |
| - | * | + | *Melanin: ampicillin, copper, and tyrosine |
| - | * | + | *Violacin: trimethoprim |
*Do 1:10 dilution with SDW into a new eppendorf | *Do 1:10 dilution with SDW into a new eppendorf | ||
*Pipette 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums | *Pipette 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums | ||
Revision as of 09:35, 29 July 2009
Categories :
Project :
-
Overview
Sensitivity Tuner
--- Characterisation
--- Modelling
Colour Generators
--- Carotenoids (Orange/Red)
--- Melanin (Brown)
--- Violacein (Purple/Green)
The Future
Safety
Notebook :
Team Logistics :
-
Protocols
Stock List
Research
Shared Links and Help
Protocols
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 culture and innoculate into larger LB volume and grow in shaking incubator until OD600 of 0.2-0.3 (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 5uL; 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 1mm separation electrocuvette = 4 total
- 5uL of biobricks
- 0.5uL of melanin and violacein plasmid
- Add 45 uL Competent cells
- Tap electrocuvette gently to evenly spread mixture in the electrocuvette gap with no air bubbles
- Thoroughly dry the cuvette
- 1.68 kV passed across cuvette, 5.1-5.4 time constant at 200 ohms and 25 uF
- Add 0.25 mL SOC liquid medium to electrocuvette
- Incubate electrocuvettes at 37 degrees C for 60 minutes
- Pipette 150uL onto a (warmed) selective LB agar plate, spread with blue spreader
- Orange genes biobrick: ampicillin
- Promoter for orange genes biobrick: ampicillin
- Melanin: ampicillin, copper, and tyrosine
- Violacin: trimethoprim
- Do 1:10 dilution with SDW into a new eppendorf
- Pipette 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums
