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

Protocol 4

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.

Protocol 5

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.

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.