Team:Calgary/Protocols

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PROTOCOLS

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Below are the protocols used by the team.

Rehydration of Registry DNA

Materials	iGEM 2018 distribution kit List Item 1 List Item 2 ddH₂O
Protocol	Add 10μL of ddH₂O to the desired well Pipette up and down 3-5 times Incubate at room temperature for 10 minutes Transform cells with 1μL of rehydrated DNA as per transformation protocol. Store the remaining amount at -20°C

Rehydration of Synthesized DNA

Materials	Synthesized DNA from IDT or Genscript ddH₂O
Protocol	Centrifuge tube containing the synthesized DNA for 1 minute at 3000g Add 20μL ddH₂O Vortex for 1 minute Incubate at 50°C for 15 minutes Briefly centrifuge. Store at -20°C

Preparation of Agar with Antibiotics

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

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 Cover flask loosely with aluminum foil, secure with autoclave tape and sterilize by autoclaving Remove agar from autoclave and allow agar to cool until warm to the touch before adding appropriate antibiotic Stir on hot plate and magnetic stirrer for 30 seconds Pour agar into plates using aseptic technique

Plating Culture Broth on Agar Plates

Materials

Luria-Bertani agar plate with appropriate antibiotic (if required) Overnight culture of desired bacteria 70% ethanol Spreading rod

Protocol

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

Preparation of Chemically Competent Escherichia coli Cells

Materials

Escherichia coli DH5α cells Luria-Bertani broth 125mm culture tubes 250mL Erlenmeyer flasks 50mL Falcon tubes, pre-chilled 1M KCl 1M MgSO4

Protocol

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

Protocol 6

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