Team:Calgary/Protocols

TOP

PROTOCOLS

---

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 DH5-α 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

Inoculate 2mL of Luria-Bertani broth with E. coli DH5-α cells and incubate overnight at 37°C, shaking at 200 rpm 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 Incubate at 37°C, shaking at 200 rpm until the subculture reaches OD600 of 0.4-0.6 Chill on ice for at least 10 minutes Spin down cells in 50mL pre-chilled Falcon tube at 2500g for 8 minutes at 4°C Resuspend cells in 10mL cold 100mM CaCl2, gently mix and place on ice for 10 minutes Centrifuge at 2500g for 8 minutes at 4°C Resuspend cells in 500μL cold 100mM CaCl2, 10% glycerol and place on ice for 10 minutes Separate cells into 50μL aliquots in pre-chilled 0.5mL microcentrifuge tubes and store at -80°C

Transformation of Chemically Competent Escherichia coli

Materials

Chemically competent E. coli DH5-α aliquots DNA for transformation Luria-Bertani broth with and without appropriate antibiotic Agar plate with appropriate antibiotic

Protocol

Thaw aliquot of competent E. coli DH5-α cells on ice Add 0.3-1μg DNA to cells (maximum 5μL), flick gently to mix, and place on ice for 45 minutes Heat shock at 42°C for 1 minute Place on ice for 5 minutes Add 250μL plain Luria-Bertani medium to aliquot of cells Incubate cells at 37°C, shaking at 200 rpm for 60 to 90 minutes Plate 100μL of resuspended culture on agar plate with appropriate antibiotic and spread Incubate plates at 37°C overnight or until desired growth is observed

Glycerol Stock Preparation of Escherichia coli

Materials	Overnight culture of transformed bacteria Sterile 1.5mL microcentrifuge tubes Sterile 50% glycerol
Protocol	Using aseptic technique, pipette 0.5mL of 50% sterile glycerol into a 1.5mL microcentrifuge tube Using aseptic technique, add 0.5 mL of overnight culture Pipette up and down gently to mix Store at -80°C

Plasmid Miniprep from Escherichia coli

Materials

Overnight culture of bacteria Resuspension buffer (stored at 4°C) 50mM Tris-HCl, pH 8 10mM EDTA 100μg/mL RNase A Lysis buffer 200mM NaOH 1% (v/v) SDS Precipitation buffer 3M CH3CO3K, pH 5.5 Isopropanol 70% ethanol, ice cold 2mL microcentrifuge tubes 1.5mL microcentrifuge tubes ddH2O

Protocol

Transfer 2mL of the overnight culture to a 2mL microcentrifuge tube and pellet the cells by spinning at 3500g for 1 minute. Discard supernatant, and repeat as necessary Resuspend pellet in 300μL Resuspension buffer Add 300μL Lysis buffer. Invert gently Quickly add 300μL Precipitation buffer. Invert gently Centrifuge at 14,000g for 10 minutes at room temperature Retain supernatant in a clean 1.5mL microcentrifuge tube Add 650μL isopropanol. Gently invert and incubate at room temperature for 10 minutes Centrifuge at 14,000g for 10 minutes at  4°C. Discard supernatant Wash pellet with 500μL cold 70% ethanol (do not resuspend) Centrifuge at 14,000g for 5 minutes at 4°C. Discard supernatant Dry pellet in vacufuge for 30 to 60 minutes Resuspend pellet in ddH2O and store at - 20°C

Restriction Digest

Materials	DNA Restriction enzymes 10X appropriate buffer ddH2O 0.2mL PCR tubes (Optional) Antarctic phosphatase (Optional) 10X Antarctic phosphatase buffer
Protocol	Add the following into a 0.2mL PCR tube: 1-3μg DNA 1μL restriction enzyme 1 1μL restriction enzyme 2 2μL 10X appropriate buffer (Optional) 2μL Antarctic phosphatase (Optional) 2μL 10X Antarctic phosphatase buffer Incubate at 37°C for one to three hours Deactivate restriction enzymes via heat shock by incubating tube at 80°C for 20 minutes

Protocol Name

Materials	Material 1 Material 2 Material 3 Sub-bullet 1 Sub-bullet 2 Sub-bullet 3
Protocol	Step 1 Step 2 Step 3

Protocol Name

Materials	Material 1 Material 2 Material 3 Sub-bullet 1 Sub-bullet 2 Sub-bullet 3
Protocol	Step 1 Step 2 Step 3