Team:Calgary/Protocols

Team:Calgary/Notebook - 2018.igem.org

PROTOCOLS



Below are the protocols used by the team.


Materials

iGEM 2018 distribution kit

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, 5g 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 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
  1. Inoculate 2mL of Luria-Bertani broth with E. coli DH5-α 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
  9. Separate cells into 50μL aliquots in pre-chilled 0.5mL microcentrifuge tubes and store at -80°C

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

Materials

Overnight culture of transformed bacteria

Sterile 1.5mL microcentrifuge tubes

Sterile 50% glycerol
Protocol
  1. Using aseptic technique, pipette 0.5mL of 50% sterile glycerol into a 1.5mL microcentrifuge tube
  2. Using aseptic technique, add 0.5 mL of overnight culture
  3. Pipette up and down gently to mix
  4. Store at -80°C

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
  1. 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
  2. Resuspend pellet in 300μL Resuspension buffer
  3. Add 300μL Lysis buffer. Invert gently
  4. Quickly add 300μL Precipitation buffer. Invert gently
  5. Centrifuge at 14,000g for 10 minutes at room temperature
  6. Retain supernatant in a clean 1.5mL microcentrifuge tube
  7. Add 650μL isopropanol. Gently invert and incubate at room temperature for 10 minutes
  8. Centrifuge at 14,000g for 10 minutes at  4°C. Discard supernatant
  9. Wash pellet with 500μL cold 70% ethanol (do not resuspend)
  10. Centrifuge at 14,000g for 5 minutes at 4°C. Discard supernatant
  11. Dry pellet in vacufuge for 30 to 60 minutes
  12. Resuspend pellet in ddH2O and store at - 20°C

Materials

DNA

Restriction enzymes

10X appropriate buffer

ddH2O

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

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
  1. Add the following to your sample, in this order
    • 1/10 volume of 3M sodium acetate
    • 2-3 volumes of 100% ethanol
  2. Mix and freeze at -80°C for 30-60 minutes
  3. Spin at 14,000 rpm for 30 minutes at 4°C
  4. Discard supernatant
  5. Dry the pellet in vacufuge for 15-30 minutes
  6. Resuspend in ddH2O and store at -20°C

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
  1. For a 1% gel, add 1g agarose to 100mL TAE buffer in a 250mL Erlenmeyer flask and microwave until agarose is fully dissolved
  2. Allow flask to cool until warm to the touch before adding 4μL RedSafe nucleic acid staining solution. Gently swirl to mix
  3. Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel
  4. Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer
  5. Load samples of DNA containing 6X loading dye
  6. Run gel at 100V for 30 minutes or until loading dye is ⅔ way down the 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
  1. 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
  2. Allow flask to cool until warm to the touch before adding 1.5μL RedSafe nucleic acid staining solution. Gently swirl to mix
  3. Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel
  4. Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer
  5. Load samples of DNA containing 6X loading dye
  6. Run gel at 80 V for 30 minutes or until loading dye is 1/2 way down the gel
  7. 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
  8. Melt the gel at 65°C for 5 minutes for ligation

Materials

Gel sample with DNA band

Spin columns

QuickClean II Gel Extraction Kit

  • Binding Buffer II
  • Wash Buffer
  • Elution Buffer, pH 7.8
Protocol
  1. Excise the DNA band from the agarose gel with a clean razorblade
  2. Weigh gel slice and add 3 volumes of binding buffer II to 1 volume of gel slice (100mg = 100μl)
  3. Incubate at 55°C for 10 minutes or until the gel slice has completely dissolved
  4. Add 1 volume of isopropanol to 1 volume of gel and mix
  5. Transfer sample to spin column, centrifuge at 6000g for 1 minute. Discard flow through until sample is fully processed
  6. Add 500μl binding buffer II to spin column, centrifuge at 12000g for 50 seconds. Discard flow through
  7. Add 750μl wash buffer to spin column, centrifuge at 12000g for 50 seconds. Discard flow through
  8. Centrifuge at 12000g for an additional 1 minute and transfer spin column from collection tube to a sterile 1.5mL microcentrifuge tube
  9. Add 30-100μl elution buffer and let it stand for 1min at room temperature
  10. Centrifuge at 12000g for 1 minute. Buffer in the microcentrifuge tube contains the DNA sample

Materials

Digested vector DNA

Digested insert DNA

10X DNA ligase buffer

T4 DNA ligase

ddH2O

0.2mL PCR tubes
Protocol
  1. 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
  2. Incubate tube at room temperature for 2 hours
  3. Use 3-10μL to transform cells, store at -20°C

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
  1. Add 4μL of PCR-grade ddH2O to 0.2mL PCR tube or 96-well plate
  2. Using aseptic technique, pick a colony and touch it with a sterile pipette tip. Place in PCR tube or 96-well plate and swirl
  3. Add 16μL cPCR mastermix to 0.2mL PCR tube or 96-well plate
  4. 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
  5. Run samples on agarose gel

Materials

Frozen HEK293T cells

100mm cell culture dishes

DMEM (Dulbecco’s Modified Eagle Medium) with 10% FBS (fetal bovine serum)
Protocol
  1. Remove media from fridge and place in 370C water bath for a minimum of 10 minutes
  2. Label a new 100mm dish properly and add 10mL of media
  3. Remove cells from -80°C and put on ice
  4. Immediately begin to thaw cells quickly in a 37°C water bath by holding the cryovial and swirling in the water
  5. Add the cells to the dish using a 1ml pipette and swirl to mix
  6. Incubate for 6-8 hours at 37°C
  7. Replace media with fresh, warm media. Check under the microscope if cells have adhered to the dish
  8. Split a few times before using in assays

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
  1. 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)
  2. Remove media, trypsin and PBS from fridge and warm in a 37°C water bath before passaging
  3. Remove media from cells
  4. Wash with 6-8ml PBS
  5. Add 2ml trypsin
  6. Incubate for 30 seconds at room temperature
  7. Add 8ml of media to the dish
  8. Wash cells off the dish with pipette
  9. Add approximately 8ml of fresh media to a new, labelled 100mm dish
  10. Add approximately 1.5ml cells to the new dish
  11. Mix the dish by swirling
  12. Incubate at 37°C

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
  1. Repeat steps 1-8 of passaging
  2. Place the cells into a 50mL falcon tube and centrifuge at 1000rpm for 5 minutes
  3. Remove the trypsin containing media and resuspend in an appropriate amount of media
  4. Count cells using a haemocytometer
  5. 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)

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
  1. 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. 2 hours before transfection change media
  3. Complete a transfection table
  4. Remove an aliquot of CaCl2 and 2x HEBS
  5. Add ddH2O to transfection tubes according to the transfection table
  6. Vortex and spin plasmids
  7. Add plasmid according to the table
  8. Add 200ul CaCl2 to each tube (diluted)
  9. Add 250ul of 2x HEBS buffer dropwise to each tube
  10. Bubble each tube 30x with an electric pipette and then vortex for 10 seconds
  11. Start a 20 minute timer after tube one is complete. Let tubes stand until timer is up
  12. Vortex 2 tubes at a time for 10 seconds and spin. Check to see if precipitate can be observed
  13. Resuspend precipitate by pipetting up and down 10x
  14. Add all of the liquid to the correct dish in a circular, clockwise manor
  15. Mix plate by shaking 3x up and down and back and forth
  16. Look at cells under the microscope to see precipitate. Precipitate should look like tiny, black dots
  17. Incubate cells for 16 hours at 37°C and 5% CO2
  18. After 16 hours change media to include antibiotic/antimitotic
  19. The next day, protein should be expressed. Assays can be performed

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

Materials

GenEluteTM kit

  1. Resuspension solution
  2. Lysis solution C
  3. Column preparation solution
  4. Wash solution
  5. Elution solution (10mM Tris-HCl, 0.5mM EDTA, pH 9.0)
  6. GenElute miniprep binding columns
  7. 2.0mL Collection tubes

Proteinase K (20mg/mL)

RNase A solution

1.5mL microcentrifuge tubes

100% ethanol
Protocol
  1. Harvest 5 x 106 cells as outlined above under "Setting of HEK293T Cells Before Transfection" until a pellet of cells is obtained
  2. Resuspend the pellet thoroughly in 200μL of resuspension solution
  3. Add 20μL of RNase A solution and incubate for 2 minutes at room temperature
  4. 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
  5. 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
  6. Add 200μL of 100% ethanol to the lysate from step 4. Mix thoroughly by vortexing for 5-10 seconds until homogenous
  7. 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
  8. 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
  9. 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
  10. 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
  11. Repeat step 10 for improved yield

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