3.3.3 Thawing of DH5α
E. coli Cell Harboring
Plasmid
1. Remove an LB agar plate containing 100 μg/mL ampicillin or
50 μg/mL kanamycin from 4
C refrigerator and warm the
plates at room temperature.
2. Remove the DH5α E. coli cells harboring plasmid from À80
C
freezer (Subheading 3.3.2, step 3), open the tube and use a
sterile loop to scrape off the frozen bacteria from the top.
3. Streak the bacteria onto the LB agar plate and incubate the
plate upside down overnight at 37
C.
4. The following day, pick the colony and inoculate into the LB
medium containing antibiotic or store the culture plate at 4
C.
3.4 Transfection
of CRISPR/Cas9
and GFP Constructs
into K562 and Raji
Cell Lines
This section is adapted from a 4D-Nucleofector™ Protocol for
K562 and Raji cells using 4D-Nucleofector™ X Unit. We perform
the transfection in 100-μL Nucleocuvette™ Vessels (see Note 8)
using the plasmids (Subheading 3.3).
3.4.1 Nucleofection
1. Change the medium for the K562 and Raji cells 24 h prior to
transfection with plasmids for CRISPR/Cas9-mediated gene
editing. Transfer the culture to a 15-mL conical tube and
centrifuge at 500 Â g for 5 min at room temperature. Discard
the supernatant and resuspend the pellet in 5 mL of culture
medium. Transfer the suspension to a 25-cm
2 cell culture flask.
2. The following day, prepare a 6-well culture plate by adding
1 mL of culture medium into a well and pre-incubate the plate
at 37
C, 5% CO 2 .
3. Prepare 100 μL of P3 Nucleofector™ Solution by adding
18 μL of Supplement to 82 μL of Nucleofector™ Solution.
Mix thoroughly by pipetting and chill the mixture on ice.
4. Harvest 1 Â 10
6 K562 or Raji cells to a 15-mL conical tube and
centrifuge at 500 Â g for 5 min at room temperature.
5. Discard the supernatant and resuspend the pellet in the P3
Nucleofector™ Solution (see Note 9).
6. Add 1 μg of each plasmid to the cell suspension and mix
thoroughly by pipetting (see Note 10).
7. Carefully transfer the mixture into the Nucleocuvette™ vessel
using the supplied pipette.
8. Gently tap the Nucleocuvette™ vessel to ensure that the sample covers the bottom of the cuvette.
9. Place the Nucleocuvette™ vessel into the retainer of the
4D-Nucleofector™ X Unit.
10. Start Nucleofection™ process by pressing “Start” on the display of 4D-Nucleofector™ Core Unit. Use the program
FF-120 for K562 cells and the program DS-104 for Raji cells.
CRISPR/Cas9-Mediated GFP Knock-in in K562 and Raji Cell Lines
223
E. coli Cell Harboring
Plasmid
1. Remove an LB agar plate containing 100 μg/mL ampicillin or
50 μg/mL kanamycin from 4
C refrigerator and warm the
plates at room temperature.
2. Remove the DH5α E. coli cells harboring plasmid from À80
C
freezer (Subheading 3.3.2, step 3), open the tube and use a
sterile loop to scrape off the frozen bacteria from the top.
3. Streak the bacteria onto the LB agar plate and incubate the
plate upside down overnight at 37
C.
4. The following day, pick the colony and inoculate into the LB
medium containing antibiotic or store the culture plate at 4
C.
3.4 Transfection
of CRISPR/Cas9
and GFP Constructs
into K562 and Raji
Cell Lines
This section is adapted from a 4D-Nucleofector™ Protocol for
K562 and Raji cells using 4D-Nucleofector™ X Unit. We perform
the transfection in 100-μL Nucleocuvette™ Vessels (see Note 8)
using the plasmids (Subheading 3.3).
3.4.1 Nucleofection
1. Change the medium for the K562 and Raji cells 24 h prior to
transfection with plasmids for CRISPR/Cas9-mediated gene
editing. Transfer the culture to a 15-mL conical tube and
centrifuge at 500 Â g for 5 min at room temperature. Discard
the supernatant and resuspend the pellet in 5 mL of culture
medium. Transfer the suspension to a 25-cm
2 cell culture flask.
2. The following day, prepare a 6-well culture plate by adding
1 mL of culture medium into a well and pre-incubate the plate
at 37
C, 5% CO 2 .
3. Prepare 100 μL of P3 Nucleofector™ Solution by adding
18 μL of Supplement to 82 μL of Nucleofector™ Solution.
Mix thoroughly by pipetting and chill the mixture on ice.
4. Harvest 1 Â 10
6 K562 or Raji cells to a 15-mL conical tube and
centrifuge at 500 Â g for 5 min at room temperature.
5. Discard the supernatant and resuspend the pellet in the P3
Nucleofector™ Solution (see Note 9).
6. Add 1 μg of each plasmid to the cell suspension and mix
thoroughly by pipetting (see Note 10).
7. Carefully transfer the mixture into the Nucleocuvette™ vessel
using the supplied pipette.
8. Gently tap the Nucleocuvette™ vessel to ensure that the sample covers the bottom of the cuvette.
9. Place the Nucleocuvette™ vessel into the retainer of the
4D-Nucleofector™ X Unit.
10. Start Nucleofection™ process by pressing “Start” on the display of 4D-Nucleofector™ Core Unit. Use the program
FF-120 for K562 cells and the program DS-104 for Raji cells.
CRISPR/Cas9-Mediated GFP Knock-in in K562 and Raji Cell Lines
223