Team:METU HS Ankara/Experiments

METU HS IGEM

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Experiments

Materials:
  • dH2O
  • iGEM Kit Plates
  • Pipette

Method:
  1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part desired.
  2. Pipette 10 µLof dH2O into the well. Pipette up and down several times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. Resuspension will be in a crimson color, as the dried DNA has crisol dye.
  3. Transform resuspended DNA into an eppendorf tube.

Competent Cell Preparation Protocol:

Buffer 1:
  • Potassium acetate 30 µL
  • RbCl 100 µL
  • CaCl 100 µL
  • 87% glycerol 4,3 mL
  • Complete to 25 mL

Buffer 2:
  • MOPS 10 µL
  • RbCl 10 µL
  • CaCl 75 µL
  • 87% glycerol 4,3 mL
  • Complete to 25 mL

Method:
  1. Pipette 100 µL of grown KO11 into 10 mL LB with 2% glucose, incubate for 2 hrs at 37 ℃ till an OD value of 0,4-0,6 is reached.
  2. Put 4,5 mL of cultures into falcon tubes, incubate on ice for 15 mins.
  3. Centrifuge at 3500 RPM at 4 ℃ for 10 mins.
  4. Discard supernatant.
  5. Resuspend pellet on ice in ice-cold 650 µL of Buffer 1.
  6. Centrifuge at 3500 RPM at 4 ℃ for 5 min.
  7. Discard supernatant.
  8. Resuspend pellet on ice in ice-cold 250 µL of Buffer 2.
  9. Aliquot as 60 µL in 1,5 mL eppendorf.
  10. Store at -80 ℃.

Overnight Culture in Falcon Tubes Protocol:

Materials:
  • 50 mL Falcon tube
  • Pipette and pipette tips
  • Incubator
  • Antibiotics
  • Cultured bacteria
  • LB broth

Method:
  1. Put 10 mL LB broth in a 50 mL falcon tube.
  2. Add 10 µL of antibiotics.
  3. Get 100 µL of cultured bacteria and add to the falcon.
  4. Place the tube in incubator for 16-18 hrs at 37℃, 135 RPM.

Competent Cell Test Kit Protocol

Materials:
  • 70% ethanol
  • Paper towels
  • Lab marker / Sharpie
  • 1,5 mL microcentrifuge tubes
  • Container for ice
  • Ice
  • Competent cell aliquot(s)
  • Competent Cell Test Kit
  • Agar plates with chloramphenicol
  • 42°C Water Bath (or hot water source and thermometer)
  • 37°C Incubators (oven and shaker)
  • LB
  • Sterile glass beads or sterile cell spreader
  • Pipette
  • Pipette tips

References