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 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, 5g 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
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 Incubate at 37°C for one to three hours Deactivate restriction enzymes via heat shock by incubating tube at 80°C for 20 minutes

Ethanol Precipitation of DNA

Materials	DNA sample that has already been digested once with the desired enzyme(s), gel purified, or DNA sample that has been isolated from HEK293T cells 3M sodium acetate, pH 5.2 100% cold ethanol ddH2O
Protocol	Add the following to your sample, in this order 1/10 volume of 3M sodium acetate 2-3 volumes of 100% ethanol Mix and freeze at -80°C for 30-60 minutes Spin at 14,000 rpm for 30 minutes at 4°C Discard supernatant Dry the pellet in vacufuge for 15-30 minutes Resuspend in ddH2O and store at -20°C

Agarose Gel Electrophoresis

Materials

TAE buffer: 40mM Tris, pH 7.6 20mM CH3COOH 1mM EDTA Agarose 250mL Erlenmeyer flask RedSafe nucleic acid staining solution Gel casting tray and comb 6X loading dye DNA sample

Protocol

For a 1% gel, add 1g agarose to 100mL TAE buffer in a 250mL Erlenmeyer flask and microwave until agarose is fully dissolved Allow flask to cool until warm to the touch before adding 4μL RedSafe nucleic acid staining solution. Gently swirl to mix Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer Load samples of DNA containing 6X loading dye Run gel at 100V for 30 minutes or until loading dye is ⅔ way down the gel

DNA Excision From Low Melting Point Gel

Materials

TAE buffer: 40mM Tris, pH 7.6 20mM CH3COOH 1mM EDTA Low melting point agarose 250mL Erlenmeyer flask RedSafe nucleic acid staining solution Gel casting tray and comb 6X loading dye Razor blade UV-safe mask and shield 1.5mL microcentrifuge tubes

Protocol

For a 1% gel, add 0.3g low melting point agarose to 30mL TAE buffer in a 250mL Erlenmeyer flask and microwave until agarose is fully dissolved Allow flask to cool until warm to the touch before adding 1.5μL RedSafe nucleic acid staining solution. Gently swirl to mix Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer Load samples of DNA containing 6X loading dye Run gel at 80 V for 30 minutes or until loading dye is 1/2 way down the gel Remove the gel from casting tray and place it on a UV-light viewer. While using a UV-protecting face mask and shield, turn on the UV light and use razor blade to excise out the desired DNA bands (as quickly as possible as UV-light damages the DNA). Place each band into individual 1.5mL microcentrifuge tubes Melt the gel at 65°C for 5 minutes for ligation

Gel Extraction Using QuickClean II Gel Extraction Kit

Materials

Gel sample with DNA band Spin columns QuickClean II Gel Extraction Kit Binding Buffer II Wash Buffer Elution Buffer, pH 7.8

Protocol

Excise the DNA band from the agarose gel with a clean razorblade Weigh gel slice and add 3 volumes of binding buffer II to 1 volume of gel slice (100mg = 100μl) Incubate at 55°C for 10 minutes or until the gel slice has completely dissolved Add 1 volume of isopropanol to 1 volume of gel and mix Transfer sample to spin column, centrifuge at 6000g for 1 minute. Discard flow through until sample is fully processed Add 500μl binding buffer II to spin column, centrifuge at 12000g for 50 seconds. Discard flow through Add 750μl wash buffer to spin column, centrifuge at 12000g for 50 seconds. Discard flow through Centrifuge at 12000g for an additional 1 minute and transfer spin column from collection tube to a sterile 1.5mL microcentrifuge tube Add 30-100μl elution buffer and let it stand for 1min at room temperature Centrifuge at 12000g for 1 minute. Buffer in the microcentrifuge tube contains the DNA sample

Ligation of DNA Inserts to Plasmid Backbones

Materials	Digested vector DNA Digested insert DNA 10X DNA ligase buffer T4 DNA ligase ddH2O 0.2mL PCR tubes
Protocol	Add to a 0.2mL PCR tube: digested vector DNA Appropriate amount of digested insert DNA to give desired insert:vector molar ratio 0.5μL T4 DNA ligase 2μL 10X T4 DNA ligase buffer ddH2O to a total volume of 20μL Incubate tube at room temperature for 2 hours Use 3-10μL to transform cells, store at -20°C

Colony PCR for Escherichia coli

Materials

Transformed bacterial colony on agar plate PCR-grade ddH2O 0.2mL PCR tubes or 96-well plate cPCR mastermix: 20μL 10X Taq polymerase buffer 4μL 10μM forward primer 4μL 10μM reverse primer 1μL 10mM Taq polymerase 127μL ddH2O

Protocol

Add 4μL of PCR-grade ddH2O to 0.2mL PCR tube or 96-well plate Using aseptic technique, pick a colony and touch it with a sterile pipette tip. Place in PCR tube or 96-well plate and swirl Add 16μL cPCR mastermix to 0.2mL PCR tube or 96-well plate Run PCR in a thermal cycler under the following conditions: Initial denaturation: 95°C for 3 minutes Denature: 95°C for 30 seconds Anneal: Tm-5°C for 30 seconds Extension: 72°C for 1 minute per kilobase Repeat denature, anneal and extension steps for 30-35 cycles Final extension: 72°C for 5 minutes Run samples on agarose gel

Thawing HEK293T Cells

Materials

Frozen HEK293T cells 100mm cell culture dishes DMEM (Dulbecco’s Modified Eagle Medium) with 10% FBS (fetal bovine serum)

Protocol

Remove media from fridge and place in 370C water bath for a minimum of 10 minutes Label a new 100mm dish properly and add 10mL of media Remove cells from -80°C and put on ice Immediately begin to thaw cells quickly in a 37°C water bath by holding the cryovial and swirling in the water Add the cells to the dish using a 1ml pipette and swirl to mix Incubate for 6-8 hours at 37°C Replace media with fresh, warm media. Check under the microscope if cells have adhered to the dish Split a few times before using in assays

Passaging and Splitting HEK293T Cells

Materials

DMEM with 10% FBS and 1% penicillin-streptomycin 0.25% Trypsin, 2.21 mM EDTA, 1X [-] sodium bicarbonate 1X PBS (phosphate buffered saline) 100mm cell culture dishes

Protocol

Look at cells in the morning each day you come to the lab. Cells should be passaged before they are fully confluent (do not allow cells to grow over 75-85% max confluence) Remove media, trypsin and PBS from fridge and warm in a 37°C water bath before passaging Remove media from cells Wash with 6-8ml PBS Add 2ml trypsin Incubate for 30 seconds at room temperature Add 8ml of media to the dish Wash cells off the dish with pipette Add approximately 8ml of fresh media to a new, labelled 100mm dish Add approximately 1.5ml cells to the new dish Mix the dish by swirling Incubate at 37°C

Setting HEK293T Cells Before Transfection

Materials

DMEM with 10% FBS and 1% penicillin-streptomycin 0.25% Trypsin, 2.21 mM EDTA, 1X [-] sodium bicarbonate 1X PBS 100mm cell culture dishes 50mL Falcon Tube Haemocytometer Tally Counter 2X HEBS (HEPES Buffered Saline)

Protocol

Repeat steps 1-8 of passaging Place the cells into a 50mL falcon tube and centrifuge at 1000rpm for 5 minutes Remove the trypsin containing media and resuspend in an appropriate amount of media Count cells using a haemocytometer Calculate the amount of cells and media/2X HEBS buffer you need to set the experiment, and set aside a little bit extra. Add the appropriate amount to the cell media mixture/2X HEBS Buffer to each plate and swirl to mix (Media is used in Calcium Phosphate Method and 2X HEBS Buffer is used in Electroporation Method)

Calcium Phosphate Method for Transfecting HEK293T Cells

Materials

DMEM with 10% FBS and 1% penicillin-streptomycin 0.25% Trypsin, 2.21 mM EDTA, 1X [-] sodium bicarbonate 1X PBS 100mm cell culture dishes ddH2O 2.5M CaCl2 2x HEBS (HEPES Buffered Saline)

Protocol

Seed the necessary number of cells in the correct plate size the day before transfection. Cells should be 25-50% confluent the next day for best transfection efficiency 2 hours before transfection change media Complete a transfection table Remove an aliquot of CaCl2 and 2x HEBS Add ddH2O to transfection tubes according to the transfection table Vortex and spin plasmids Add plasmid according to the table Add 200ul CaCl2 to each tube (diluted) Add 250ul of 2x HEBS buffer dropwise to each tube Bubble each tube 30x with an electric pipette and then vortex for 10 seconds Start a 20 minute timer after tube one is complete. Let tubes stand until timer is up Vortex 2 tubes at a time for 10 seconds and spin. Check to see if precipitate can be observed Resuspend precipitate by pipetting up and down 10x Add all of the liquid to the correct dish in a circular, clockwise manor Mix plate by shaking 3x up and down and back and forth Look at cells under the microscope to see precipitate. Precipitate should look like tiny, black dots Incubate cells for 16 hours at 37°C and 5% CO2 After 16 hours change media to include antibiotic/antimitotic The next day, protein should be expressed. Assays can be performed

Electroporation Method for Transfecting HEK293T Cells

Materials

DMEM with 10% FBS and 1% penicillin-streptomycin 0.25% Trypsin, 2.21 mM EDTA, 1X [-] sodium bicarbonate 1X PBS 100mm cell culture dishes ddH2O 2x HEBS (HEPES Buffered Saline) Lonza Cuvettes (Amaxa nucleofector specific brand) RNP (CRISPR/Cas9 wild-type or nickase) complexed with sgRNA (nickase requires a pair of sgRNAs)

Protocol

Harvest 5 x 106 cells as outlined above under "Setting of HEK293T Cells Before Transfection" Resuspend cells in 20μL of 2X HEBS buffer Add 5μL of RNP/sgRNA solution, mix, and add to a cuvette Tap lightly to remove air bubbles Use program Q-100 on an Amaxa nucleofector Add electroporated cells to 75μL of pre-warmed media and resuspend the cells Transfer 25μL of resuspended cells to 175μL of culture media (DMEM with 10% FBS) Incubate cells in a tissue culture incubator (37°C, 5% CO2) for 72 hours

Genomic DNA Extraction From HEK293T Cells Using GenElute Mammalian Genomic DNA Miniprep Kit

Materials

GenEluteTM kit Resuspension solution Lysis solution C Column preparation solution Wash solution Elution solution (10mM Tris-HCl, 0.5mM EDTA, pH 9.0) GenElute miniprep binding columns 2.0mL Collection tubes Proteinase K (20mg/mL) RNase A solution 1.5mL microcentrifuge tubes 100% ethanol

Protocol

Harvest 5 x 106 cells as outlined above under "Setting of HEK293T Cells Before Transfection" until a pellet of cells is obtained Resuspend the pellet thoroughly in 200μL of resuspension solution Add 20μL of RNase A solution and incubate for 2 minutes at room temperature Add 20μL of Proteinase K solution to the sample, followed by 200μL of lysis solution C. Vortex thoroughly for 15 seconds and incubate at 70°C for 10 minutes Assemble a binding column with a 2mL collection tube. Add 500μL of column preparation solution to the binding column and centrifuge at 12000g for 1 minute. Discard flow through liquid, but retain the collection tube Add 200μL of 100% ethanol to the lysate from step 4. Mix thoroughly by vortexing for 5-10 seconds until homogenous Transfer tube contents into the treated binding column from step 5. Centrifuge at >6500g for 1 minute. Discard the collection tube containing the flow through liquid and place the binding column in a new 2mL collection tube Add 500μL of wash solution to the binding column and centrifuge for 1 minute at >6500g. Discard the flow through liquid, but retain the collection tube Add another 500μL of wash solution to the binding column, centrifuge for 3 minutes at maximum speed (12000-16000g) to dry the binding column. Discard the collection tube containing the flow through liquid and place the binding column in a new 2mL collection tube Pipette 200μL of the elution solution directly onto the center of the binding column and incubate for 5 minutes, centrifuge for 1 minute at 6500g to elute the DNA Repeat step 10 for improved yield

Freezing HEK293T Cells

Materials

DMEM with 10% FBS and 1% penicillin-streptomycin 0.25% Trypsin, 2.21 mM EDTA, 1X [-] sodium bicarbonate 1X PBS 100mm cell culture dishes 10% DMSO Cryovials Cryocontainer 50mL Falcon Tube Isopropanol

Protocol

Prepare the required amount of chilled (at 4°C) 10% DMSO in DMEM with 10% FBS and 1% penicillin-streptomycin media Label the correct number of cryovials with cell name, passage number and date, dish size to which the cells in the vial should be thawed Remove media, trypsin and 1X PBS from fridge and warm in a 37°C water bath before freezing Wash with 6ml 1X PBS Add 2ml trypsin Incubate for 30 seconds at room temperature or at 37°C Add 8ml of media to the dish Wash cells off the dish with pipette Place the cells into a falcon tube and centrifuge at 1000rpm for 5 minutes Remove the media To the pellet add appropriate amount of chilled 10% DMSO containing media Resuspend the pellet well. Add 0.5ml to each cryovial. Divide the remaining liquid in the cryovials (no need to be accurate) Place the cryovials in a cryocontianer containing room temperature isopropanol and put in -80°C overnight Next day, remove the cryovials freeze the vials in liquid nitrogen storage. The vial can be stored in -80°C freezer

Strawberry DNA Extraction

Materials	Strawberries Ziploc bag Strawberry DNA Extraction Buffer 10mL detergent 90mL water 1.5g of NaCl Coffee filter Beaker or plastic cup Ethanol Stir rod or toothpick
Protocol	Place two strawberries into a Ziploc bag and squish until smooth Pour 20mL of Strawberry Extraction Buffer into the Ziploc bag Seal the bag and gently mix contents Pour contents of bag through coffee filter into beaker or cup Pour 10mL of ethanol gently down side of cup Incubate for 30 seconds at room temperature or at 37°C Swirl in between ethanol and water layer in cup

Strawberry Daiquiri

Materials	Frozen strawberries Pineapple juice Alcohol (must be 80-proof) Mesh or filter Ziploc bag
Protocol	Place 250g of strawberries and 75g of pineapple juice in a bag and gently crush with fingers Heat bag at 60°C for 10 minutes Ice bag for 10 minutes Strain pulp through mesh Wash pulp with 10mL of alcohol