Generation of HeLa KO cells with CRISPR

• Day1: thawed HeLa cells and kept them in DMEM + 1% P/S + 10% FCS
• Day 3: splitted them 1: 10 (3x) and 1:5 (3x)
• Day 5: prepared 5x 10 cm dishes with 1Mio cells each and 10x10 cm dishes with 0.5 Mio cells each
  • cell counts: A: 3.5x10^5 cells/mL, B: 3.2x10^5 cells/mL, Average: 3.35x10^5 cells/mL
  • had ca. 10 Mio cells in total → 1.5 mL = 0.5 Mio cells; 3 mL = 1 Mio cells
• Day 6: collect medium of the cells (don´t trash) and sterile filter for preparation of conditioned medium later on
  • transfection of dishes with 0.5 Mio cells per dish (each condition twice)
    • 2 µg of pX458 vector  → add to 500 µl OptiMEM
    • add respective amount of FuGENE without touching the tube wall with the tip (FuGENE: DNA ratio → 3:1)
    • vortex shortly and incubate 15 min at RT
    • add dropwise to the cells
  • dishes with 1 Mio cells were already confluent → splitted them again 1:3 and prepared another 10 cm dishes
  • transfection was performed in the later afternoon/ evening as described above
• Day 7: Change to fresh medium to remove transfection reagent
• Day 9: FACS! Make appointment early enough.
  • Preparation of conditioned medium (20%)
  • prepare 4x 96 well plates with 20% conditioned medium per construct (100 µl per well)
  • store plates with medium at 37°C, 5% CO₂ until needed
  • trypsinize cells
    • wash with 10 ml PBS
    • 1 ml trypsin per dish → 1min @ RT
    • stop with 9 ml medium
    • spin down: 4 min, 100xg
    • resuspend  pellet in HBSS (1ml) w/o MgCl₂, w/o CaCl₂ and w/o phenol red
    • add another 1ml of HBSS and use cell strainer (blue, 40 µm) to get single cells in a 50 ml tube, wash strainer with 1ml HBSS
    • transfer cells to FACS tube → transport tubes and prepared 96-well plates in a styrofoam box to the FACS facility
  • FACS sorting: Aria 2 → tell FACS facility guy what you want to do and he will calibrate the FACS machine for 96 well format
  • store cells at 37°C, 5% CO₂ after FACS sorting
• Day 16: added 100 µl of fresh medium to 96 well plates (gently on top of existing medium, to not disturb the clones too much)
• Day 20: counted clones
  • on average 15-20 clones per plate in our hands
• Day 22: changed medium
• Day 24: split clones that are already big enough on to 3x 96-well plates
• Day 27: freeze down 2 96-well plates
  • wash with PBS
  • add 30 µl of trypsin, incubate 6 min
  • add 70 µl of DMEM
  • pipette up and down 3x
  • add 100 µl freezing medium (30 ml FCS + 10 ml DMSO + 10 ml DMEM)
  • wrap in paper, put in styrofoam box, put at -80°C
• Day 28: Preparation of DNA from cells in 96-well plates
  • aspirate medium
  • wash 1x with PBS
  • add 100 µl lysis buffer (with freshly added Proteinase K)
    • 10 mM Tris-HCl, pH 8.0 (1M stock → 5 ml for 500 ml)
    • 10 mM EDTA (0.5 M stock → 10 ml for 500 ml)
    • 0.2% SDS (10% SDS stock → 10 ml for 500 ml)
    • 100 mM NaCl (5 M stock → 10 ml for 500 ml)
    • 0.2mg/ml Proteinase K (20mg/ml stock → 500 µl for 50 ml) add freshly!!!
  • incubate 6h at 55°C in humid chamber (plastic bag with wet paper)  use incubator w/ shaker
  • add 10 µl 8M LiCl and 100 µl 100 % Isoprop
  • incubate o/n on shaker in humid chamber at RT (the longer the incubation the better the yield)
• Day 29: day 2 of DNA prep
  • centrifuge 20 min at 3500 rpm, 4°C, 96-well plates will break at this speed (clean centrifuge)
  • discard supernatant (by tapping)
  • wash once with ice-cold 70% EtOH (pure, prechilled at -20°C)
  • centrifuge 10 min at 3500 rpm, 4!C
  • discard supernatant (by tapping)
  • dry plate (RT), do not overdry (→ if it does not smell like EtOH it´s good)
  • dilute DNA o/n in 50 µl low TE in humid chamber (55°C)
    • 10 mM Tris-HCl, pH 8.0
    • 0.1 mM EDTA (1mM in TE)  autoclaved or sterile filtered
• Day 30: Sequencing PCR
  • gel purification → elution in 20 µl ddH2O → use 5 µl per sequencing reaction

Alternatively to freezing down cells in the 96-well plate format, you may also freeze them in cryotubes in liquid nitrogen – much more work, but like that it is much more unlikely that you will lose good clones due to them not surviving the recovery from the 96-well-plate at -80°C. However, the DNA prep for genotyping should absolutely be done in the 96-well format via LiCl/isoprop as it is robust, cheaper and much less work than e.g. extracting DNA via a kit (columns)

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