Nov 20, 2025
Genome Edited (KO) Cell Line Generation – PCR Validation
Forked from Genome Edited (KO) Cell Line Generation –
- Amanda Bentley-DeSousa1,2,3,4,5,
- Devin Clegg1,2,3,4,5,
- Shawn Ferguson1,2,3,4,5,6
- 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 2Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 3Program in Cellular Neuroscience, Neurodegeneration and Repair;
- 4Wu Tsai Institute Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
- 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Protocol Citation: Amanda Bentley-DeSousa, Devin Clegg, Shawn Ferguson 2025. Genome Edited (KO) Cell Line Generation – PCR Validation. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl88wj6l2w/v1
Manuscript citation:
https://doi.org/10.1101/2023.10.31.564602
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 01, 2025
Last Modified: November 20, 2025
Protocol Integer ID: 228804
Keywords: RAW 264.7, KO, Synthego, ko cell lines in raw, ko cell line, cell line generation, cell lines in raw, genome edited, genome, cell, line, ko, raw, pcr validate ko cell line, pcr validate ko cell lines in raw, pcr validation, pcr validation this protocol details step
Funders Acknowledgements:
Aligning Science Across Parkinson’s Disease
Grant ID: ASAP-025173
Abstract
This protocol details steps taken to create and PCR validate KO cell lines in RAW 264.7 cells.
Materials
DMEM (Thermo Fisher Scientific, 11965-092)
FBS (Thermo Fisher Scientific, 16140-071)
Penicillin/Streptomycin (Thermo Fisher Scientific, 15140122)
CellStripper (Corning, 356230)
Synthego CRISPR Gene Knockout V2 Mouse Kits
Lipofectamine CRISPRiMAX (ThermoFisher Scientific, CMAX00003)
Cas9 (Synthego CRISPR Gene Knockout V2)
Opti-Mem (Thermo Fisher Scientific, 31985062)
Troubleshooting
Day 0
Plate 2.5 x 10^5 RAW 264.7 cells per well in a 6-well dish.
Day 1
Transfect cells using Lipofectamine CRISPRiMAX, gene specific sgRNAs and recombinant Cas9 (Synthego CRISPR Gene Knockout V2).
CRISPRiMAX protocol
In Tube 1 combine 62.5 µL Opti-MEM, 3.25 µL sgRNA (from 3 uM stock), 2.5 µL Cas9 (from 3 uM stock), and 2.5 µL Cas9+ then incubate for 00:05:00 . In Tube 2 combine 62.5 µL Opti-MEM and 3.75 µL CRISPRMAX then incubate for 00:05:00 . Combine tube 1 and tube 2. Incubate for 00:20:00 . Add drop-wise to 1 well containing 1.8 mL of media.
Day 3
Change the media.
Daty 4
Plate single cells into 96-well dishes to obtain clonal cell lines. After subculturing, confirm cell lines via immunoblotting
Expand clonal cell lines to 24-well dish
(Design primers flanking Cas9 cut sites)
Harvesting DNA using Quick Extract
8m 20s
Dissociate cells from 24-well dish
Centrifuge Cells 1000 rpm, 00:03:00
Discard supernatant
Add 200 µL of Quick Extract to cells
Vortex sample for 00:00:10
10s
Incubate sample at 65 °C for 00:06:00
6m
Vortex sample for 00:00:10
10s
Incubate sample at 98 °C for 00:02:00
2m
PCR Validation
33m
Run PCR using designed primers
PCR Reaction Setup
GoTaq‱ Green Master Mix, 2X 12.5 µL
upstream primer, 10µM 1 µL
downstream primer, 10µM 1 µL
DNA template3 µL
Nuclease-free water 7.5 µL
Total 25 µL
Thermocycler:
Initial denaturation 98 °C 00:02:00
denaturation 98 °C 00:00:30
Annealing 00:00:30 Temp. is determined by primers
Extension 72 °C 1 minute per kilobase
30 Cycles
Hold 4 °C
3m
Run PCR reactions on 1% agarose gel
100v for 00:30:00
30m