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 cultture and innoculate into larger LB volume and grow in shaking incubator until OD600 of 1.5 (4 hours?)
+
*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 2L; the rest needs to go in an eppendorf, labeled with biobrick number, and stored at -20 degrees C  
+
**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 2mm separation electrocuvette = 4 total
+
*Vector DNA pipetted into chilled 1mm separation electrocuvette = 4 total
-
*2uL of biobricks
+
*5uL of biobricks
-
*? uL of melanin and violacein plasmid
+
*0.5uL of melanin and violacein plasmid
-
*Add 45 uL Competent cells (Duncan’s suggestion was 0.5L DNA to 40 L 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 (need to ask James exactly what voltage) passed across cuvette, 5.1-5.4 time constant at 200 ohms and 25 F
+
*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 (Duncan suggests 100L) to electrocuvette  
+
*Add 0.25 mL SOC liquid medium to electrocuvette  
-
*Transfer culture to chilled eppendorf, WAIT 2 MINUTES
+
*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 200L onto a selective LB agar plate, spread with blue spreader, do duplicates, 4 separate inoculums
+
*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 NOT MADE YET
+
*violacin: trimethoprim  
*Do 1:10 dilution with SDW into a new eppendorf
*Do 1:10 dilution with SDW into a new eppendorf
-
*Pippete 200L onto a selective LB agar plate, spread with blue spreader do duplicates, 4 separate inoculums
+
*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
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