How We Validate Knockout Clones: PCR, Sanger and Western Blot

Each of the three methods answers a different question, and each has a blind spot that the others cover:

The combination is not redundancy — it is coverage. PCR without sequencing reports a deletion you have not characterised; sequencing without protein data reports a genotype you have not connected to function.

1. Junction PCR across both breakpoints. Fast, and eliminates clones where the deletion did not happen. Primers are placed outside the breakpoints so a wild-type allele still amplifies.

2. Sanger sequencing of the junction product. Confirms the breakpoint positions and reveals indels introduced during repair. Clones with unexpected junctions are re-amplified with a second primer pair before being rejected.

3. Zygosity by digital PCR or quantitative junction analysis. Distinguishes homozygous from heterozygous clones. Heterozygous clones are retained only when the project calls for them.

4. Western blot with a validated antibody. Confirms protein absence. Where the antibody is uncertain, a positive-control lysate and a loading control are run on the same membrane.

5. Optional: transcript-level check. Where the gene has alternative start sites or exons, an RT-PCR across the transcript confirms no shorter product survives.

The fourth rule is the one that saves projects: a homozygous junction with detectable protein means the model is not a knockout, regardless of what the genotype looks like.

• • Junction sequence, both breakpoints, with primer sequences
• • Zygosity and the assay used
• • Western blot image with loading and positive controls
• • Passage number at freezing, and the passage used for validation
• • A matched non-targeting or parental control from the same campaign

That last item matters for interpretation: comparing an edited clone against a parental line that never went through editing conflates the edit with the process.

Every knockout line we deliver carries the five-item package above, and we will share the raw sequencing and blot images rather than conclusions alone.

Tell us the gene and the cell type. Contact EDITGENE to request a project consultation or quote.

Validation confirms the clone at the passage tested; continued culture can change it, and re-verification at the passage of use remains advisable.

Antibody performance varies; where a validated antibody is unavailable, this is stated rather than substituted silently.

• • Sentmanat MF, et al. A survey of validation strategies for CRISPR-Cas9 editing. Scientific Reports. 2018;8:888.
• • Brinkman EK, et al. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Research. 2014;42:e168.
• • Geraghty RJ, et al. Guidelines for the use of cell lines in biomedical research. British Journal of Cancer. 2014;111:1021–1046.