Team:Calgary/Protocols

Team:Calgary/Notebook - 2018.igem.org

PROTOCOLS



Below are the protocols used by the team.

Materials

iGEM 2018 distribution kit

  • List Item 1
  • List Item 2

ddH₂O
Protocol
  1. Add 10μL of ddH₂O to the desired well
  2. Pipette up and down 3-5 times
  3. Incubate at room temperature for 10 minutes
  4. Transform cells with 1μL of rehydrated DNA as per transformation protocol. Store the remaining amount at -20°C

Materials

Synthesized DNA from IDT or Genscript

ddH₂O
Protocol
  1. Centrifuge tube containing the synthesized DNA for 1 minute at 3000g
  2. Add 20μL ddH₂O
  3. Vortex for 1 minute
  4. Incubate at 50°C for 15 minutes
  5. Briefly centrifuge. Store at -20°C

Materials

Luria-Bertani broth with agar:

  • 10% (w/v) tryptone
  • 5% (w/v) NaCl
  • 10% (w/v) yeast extract
  • 15% (w/v) agar

Appropriate antibiotic:

  • Ampicillin (final concentration of 100μg/mL)
  • Chloramphenicol (final concentration of 25μg/mL)
  • Kanamycin (final concentration of 50μg/mL)

dH₂O

1500mL Erlenmeyer flask

Stir bar

Aluminum foil
Protocol
  1. In a 1500mL Erlenmeyer flask, add 10g tryptone, 5 g yeast extract, 10g NaCl and 15g agar. Dissolve solids in 1000mL dH₂O and add a stir bar
  2. Cover flask loosely with aluminum foil, secure with autoclave tape and sterilize by autoclaving
  3. Remove agar from autoclave and allow agar to cool until warm to the touch before adding appropriate antibiotic
  4. Stir on hot plate and magnetic stirrer for 30 seconds
  5. Pour agar into plates using aseptic technique

Materials

Luria-Bertani agar plate with appropriate antibiotic (if required)

Overnight culture of desired bacteria

70% ethanol

Spreading rod
Protocol
  1. Using aseptic technique, pipette 100μL of bacterial culture onto agar plate
  2. Dip spreading rod in 70% ethanol, pass over flame, and allow for excess liquid to burn off. Cool rod on agar, avoiding bacterial culture
  3. Use rod to spread bacterial culture over entire plate, spinning the plate at the same time
  4. Dip spreading rod in 70% ethanol, pass over flame, and allow excess liquid to burn off
  5. Incubate plates at 37°C overnight or until growth is observed

Materials

Escherichia coli DH5α cells

Luria-Bertani broth

125mm culture tubes

250mL Erlenmeyer flasks

50mL Falcon tubes, pre-chilled

1M KCl

1M MgSO4

100mM CaCl2

100mM CaCl2, 10% glycerol

0.5mL microcentrifuge tubes, pre-chilled
Protocol
  1. Inoculate 2mL of Luria-Bertani broth with E. coli DH5α cells and incubate overnight at 37°C, shaking at 200 rpm
  2. Subculture 1mL of the E.coli overnight culture in 49mL fresh Luria-Bertani broth in a 250mL Erlenmeyer flask with 500μL 1M MgSO4 and 50μL 1M KCl
  3. Incubate at 37°C, shaking at 200 rpm until the subculture reaches OD₆₀₀ of 0.4-0.6
  4. Chill on ice for at least 10 minutes
  5. Spin down cells in 50mL pre-chilled Falcon tube at 2500g for 8 minutes at 4°C
  6. Resuspend cells in 10mL cold 100mM CaCl2, gently mix and place on ice for 10 minutes
  7. Centrifuge at 2500g for 8 minutes at 4°C
  8. Resuspend cells in 500μL cold 100mM CaCl2, 10% glycerol and place on ice for 10 minutes

Experimental Details

Registry DNA was rehydrated for completion of the Interlab Study. Also, Part:BBa_K934001 (phaC1-A-B1) was rehydrated and transformed into our chassis so that PHB was produced and preliminary secretion assays could be performed before the Synthesis subgroup had completed their cloning.
Materials
  • iGEM 2017 distribution kit
  • ddH₂O
Protocols
  1. Add 10μL of ddH₂O to the desired well.
  2. Pipette up and down 3-5 times.
  3. Incubate at room temperature for 10 minutes.
  4. Transform cells with 1μL of rehydrated DNA. Store the remaining amount at -20°C.