Team:WPI Worcester/Protocols


Notebook

Counting Cells

  1. Split cells if necessary
  2. Pipette 10 μL of cells into microcentrifuge tube
  3. Add 10 μL of trypan blue to the microcentrifuge tube (mix together by pipetting up and down)
  4. Wipe down counting grid and cover with ethanol
  5. Take 10 μL of cell and trypan blue mixture and place in grid (make sure there are no bubbles and the mixture spreads over the grid)
  6. Check under the microscope and count cells in corner boxes and center box
  7. Cell count should be between 100 and 500 cells
  8. Multiply total cells by 4000 to get number of cells/mL
  9. Multiply the number of wells plus one by the concentration you want
  10. Divide the number found in #8 by the number of cells/mL (found in #7) to find the amount of cell mixture needed
  11. Subtract the amount of cell mixture needed (answer from #9) from the number of wells plus one to find the amount of media needed
  12. Create the correct mixture needed from your calculations
  13. Pipette 1 mL into each well
  14. Incubate

DMEM Media:

100 mL of Media Composition

  1. 88% DMEM - 88 mL
  2. 10% FBS - 10mL
  3. 1% Glutamine - 1mL
  4. 1% PS - 1ml

eGFP ACG/ATG Transfection:

12 Well DNA Transfection Protocol for each 3 well column

  1. CaPhos-DNA Mix
    1. 20 μL 2.5 M CaCl2
    2. 4 μg total DNA (3 μg eGFP DNA, 1 ug RFP)
      1. GFP: Use either pEGFPN3 -> 3 μg = 4.62 μL or pEGFPC1 -> 3 μg = 3.42 μL or ATGeGFP -> 3 μg = 4.47 μL or ACGeGFP -> 3 μg = 4.2 uL
      2. RFP: Use PTagRFPC -> 1 μg = 1.14 μL
    3. Dilute to 200 μL with 0.1X TE
    4. Pipet up and down to mix
  2. Add 1 equal volume (200 μL) of 2X HBS solution
  3. Pipet up and down to mix (volume of tube is now 400 uL)
  4. Incubate at room temp for 1 min
  5. Add carefully 100 μL each to 3 wells (this will be 1 μg total DNA per well)
  6. Incubate for 2-6 hours in 37 degree incubator

Mammalian Cell Transfection

  1. Add 50µL 2.5M CaPhos to a 1.5mL eppendorf tube
  2. Add a total of 10µg DNA (5µg RFP, 5µg GFP)
  3. Add 0.1xTE to a total volume of 500µL
  4. Add 500µL 2xHBS, pipette up and down to mix, sit for about 1 minute
  5. Add appropriate amount to each well, do not mix

Microscopy:

Fixing Cells

    Steps 1-3 should be done in the sterile culture hood

  1. Aspirate off media and replace with 1x PBS (remove media and add PBS slowly from the side of the wells as not to disrupt cells)
  2. Aspirate off PBS
  3. Add 0.5 mL of 4% PFA (in 1x PBS) (this is the fixing agent)
  4. Put cells on a shaker with a non-transparent cover (to prevent bleaching of the biomarkers) for 10 minutes since the addition of the PFA

    5. Steps 5-7 should be done inside the chemical fume hood

  5. Remove as much PFA from the wells and put in special waste bottle
  6. Rinse with PBS and discard that into same waste bottle
  7. Add about 1 mL of PBS into each well (be sure to not add directly to well to not disturb cells
  8. Place wells under cover until they are mounted

Mounting Cells

  1. Add one drop of polyvinyl mounting solution (specially made) on glass slide for each coverslip of cells being placed on the slide
  2. Use tweezers to extract coverslip out of well, dab side of slip on paper towel to get off excess PBS, and place slip cell side down onto the drop of mounting solution
    1. Press slip gently onto mounting solution to get rid of air bubbles
  3. Use kim wipe and firmly press straight down onto slide with slips to squeeze out excess mounting solution (be sure to press evenly and try not to move slip around when pressing)
  4. Spray DI water against slide around the coverslips and press again with a kimwipe to remove excess mounting solution
  5. Cover from light to prevent bleaching, and let dry for about an hour

Miniprep:

  1. Spin liquid culture at 3,000 rpm for 10 min
  2. Discard the liquid
  3. Add 250 µL A1 to re-suspend the cells
  4. Draw up all the cells, and place in a centrifuge tube
  5. Add 250 µL A2, mix
  6. Add 300 µL A3
  7. When the color is gone, spin at max speed for 10 min
  8. Load supernatant in a column places over another tube
  9. Spin at max speed for 1 min. Discard the liquid
  10. Add 600 µL A4, spin for 30s
  11. Discard the liquid, and spin for 1 min
  12. Place the column in a new centrifuge tube, add 50 µL elution buffer, and let rest at RT for 1 min
  13. Spin for 1 min

PCR:

PCR Mixture:

  1. 22.5 µL ddH20
  2. 1.25 µL 10nM Primer (3’)
  3. 1.25 µL 10nM Primer (5’)
  4. 100 ng/µL DNA Template
  5. 25 µL 2x PCR Master Mix

PCR Protocol:

  1. 95 ℃ 2 minutes
  2. 95 ℃ 30 seconds
  3. 50 ℃ 45 seconds
  4. 72 ℃ 4.5 minutes
  5. Repeat 1-4 for 30 cycles
  6. 72 ℃ 10 minutes
  7. Hold at 10 ℃ until needed

Splitting Cells

Splitting Cells of Adherent Cell Line

  1. Aspirate off old media from culture
  2. Add 3-4 mL of PBS
    1. Swirl around PBS to cover all of the flask
  3. Aspirate off PBS
  4. Add 0.5 mL of Trypsin
    1. Swirl around the flask and lightly strike the side of the flask to remove the cells from the flask (should see the cells sliding off the flask)
  5. Add 6 mL of media to the flask and mix the solution by pipetting the media up and down
    1. Doing so can break apart clumps of cells and allow for more even distribution
  6. Place mixture into a sterile 15mL conical tube
  7. Add required amount of cells and media needed for degree of split required to have a final volume of 6 mL into a new 250 mL flask (or to the original flask)

Test Digest:

One Enzyme:

  1. Add 16 µL deionized autoclaved H20
  2. 2 µL CutSmart Buffer
  3. 1 µg pRetro-ON (calculate volume needed using concentration)
  4. 1 µL Enzyme
  5. Incubate 1-2 hours or overnight

Two Enzyme:

  1. Add 15 µL deionized autoclaved H20
  2. 2 µL CutSmart Buffer
  3. 1 µg pRetro-ON (calculated volume needed using concentration)
  4. 1 µL First Enzyme
  5. 1 µL Second Enzyme

Control:

  1. 17 µL deionized autoclaved H20
  2. 2 µL Cutsmart Buffer
  3. 1 µg pRetro-ON (calculated volume needed using concentration)

Thawing Cells Protocol:

  1. 5ml media then draw up the cell line into the same pipette
  2. Slowly dispense into final tube. Mix lightly, let rest for a few minutes
  3. Run through centrifuge at 800 RPM for 5 minutes
  4. Pipette as much of it as you can off without disturbing the pellet of cells at the bottom of the tube
  5. Dispose of old media in sterilized beaker
  6. Flick the bottom of the tube to re-suspend the cells
  7. Use approximately 6ml of media in the new flasks
  8. Gently add and slowly pipette up and down in the tube. Pipette into the flask.
  9. Label using ethanol resistant pen

Transformation:

  1. Centrifuge DNA sample at a low speed for about 1 minute
  2. Add 50µL of cells and 1µL DNA to a 1.5mL eppendorf tube
  3. Incubate the mixture in ice for 30 minutes
  4. Heat shock the mixture by placing the tube into a 42℃ water bath for 45-60 seconds
  5. Add 200 LB recovery media to the mixture
  6. Incubate on a rotating/shaking incubator for 1-2 hours
  7. Plate

Gibson Assembly Transformation Protocol:

  1. Set Up
  2. 1.) Set up reaction on ice
  3. -100 ng vector -> pRETRO-ON (cut with Not1 and BamH1)
  4. -150 ng dCas Insert
  5. -200 ng APOBEC Insert
  6. -10 ul Mastermix
  7. -Bring up to 20ul Total Volume with Water
  8. 2.) Place in PCR machine set at 50 degrees Celsius infinity for 30 minutes
  9. 3.) Store at –20 degrees Celsius until ready for Gibson transformation protocol

Transformation Protocol:

  1. Thaw competent cells on ice
  2. Add 2ul of chilled assembly product to 50 ul DH5(alpha) cells
  3. Place mixture on ice for 30 mins
  4. Heat shock for 30 seconds at 42 degrees Celsius
  5. Transfer tubes to ice for 2 mins
  6. Add 950 ul of room-temperature SOC to tube
  7. Incubate at 37 degrees Celsius for 60 minutes and shake
  8. Warm selection plates to 37 degrees Celsius
  9. Spread 100 ul of cells onto selection plates (using AMP for negative control)
  10. Incubate overnight at 37 degrees Celsius