Team:Cambridge/Protocols
From 2009.igem.org
(Difference between revisions)
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Starting from a single colony on a plate: | Starting from a single colony on a plate: | ||
*Transfer colony into 50ml liquid LB media and leave in a 200rpm shaking incubator overnight | *Transfer colony into 50ml liquid LB media and leave in a 200rpm shaking incubator overnight | ||
- | *Take 10ml of the | + | *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 | + | *Put culture on ice for 30 minutes |
*Centrifuge at 4000g for 6 minutes | *Centrifuge at 4000g for 6 minutes | ||
*Remove supernatent and resuspend cells in an equal volume of ice-cold 0.1mM HEPES | *Remove supernatent and resuspend cells in an equal volume of ice-cold 0.1mM HEPES | ||
Line 25: | Line 25: | ||
**With pipette tip, punch hole through foil cover into designated well | **With pipette tip, punch hole through foil cover into designated well | ||
**Add 20uL DIW | **Add 20uL DIW | ||
- | **We will be removing about | + | **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 | *Violacein and melanin need to be thawed | ||
- | *Vector DNA pipetted into chilled | + | *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 | + | *Add 45 uL Competent cells |
*Tap electrocuvette gently to evenly spread mixture in the electrocuvette gap with no air bubbles | *Tap electrocuvette gently to evenly spread mixture in the electrocuvette gap with no air bubbles | ||
*Thoroughly dry the cuvette | *Thoroughly dry the cuvette | ||
- | *1.68 kV | + | *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 | + | *Add 0.25 mL SOC liquid medium to electrocuvette |
- | + | *Incubate electrocuvettes at 37 degrees C for 60 minutes | |
- | *Incubate at 37 degrees C for 60 minutes | + | *Pipette 150uL onto a (warmed) selective LB agar plate, spread with blue spreader |
- | *Pipette | + | |
*orange genes biobrick: ampicillin | *orange genes biobrick: ampicillin | ||
*promoter for orange genes biobrick: ampicillin | *promoter for orange genes biobrick: ampicillin | ||
*melanin: ampicillin, copper, and tyrosine | *melanin: ampicillin, copper, and tyrosine | ||
- | *violacin: trimethoprim | + | *violacin: trimethoprim |
*Do 1:10 dilution with SDW into a new eppendorf | *Do 1:10 dilution with SDW into a new eppendorf | ||
- | *Pippete | + | *Pippete 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums |
Revision as of 11:26, 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 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
- Pippete 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums