Apr 20, 2026
Genotyping Atp13a4(KO) mice
- 1KU Leuven;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Sarah van Veen, Peter Vangheluwe 2026. Genotyping Atp13a4(KO) mice. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3q8kev25/v1
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: December 20, 2024
Last Modified: April 26, 2026
Protocol Integer ID: 116568
Keywords: ASAPCRN, genotyping, mouse, ATP13A4, dna extraction, step process for dna extraction, pcr amplification, mouse biopsy, using pcr, knockout genotype, dna, mice this protocol, genotyping, ear biopsy
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol outlines the step-by-step process for DNA extraction from mouse biopsies and genotyping of Atp13a4(KO) mice using PCR. DNA is extracted from tail or ear biopsies using a simple alkaline lysis method followed by neutralization. PCR amplification is performed using specific primers for Atp13a4, and products are analyzed on an agarose gel to distinguish between wild-type and knockout genotypes.
Materials
- Atp13a4(KO) mouse strain, C57BL/6NJ-Atp13a4em1(IMPC)Bay/Mmnc (RRID:MMRRC_050715-UNC)
- gDNA isolated from ear/tail biopsies
- Genotyping mix: PCR Bio Rapid (2x)
- PCR tubes
- UltraPure‱ Agarose (Invitrogen; cat no 16500-500)
- Midori Green Nucleic Acid Staining Solution (Bulldog-bio; cat no MG04: 10 000X)
- GeneRuler 100 bp DNA Ladder (VWR; cat no 733-2602)
DNA extraction from mouse biopsies
10m
Put a small piece of ear in a clean Eppendorf tube.
While gently opening the tube, make sure the sample is still inside and add 90 µl of 50mM NaOH and make sure the sample is inside the liquid. If the sample is not fully immersed spin it down (table top centrifuge).
Incubate this mixture for 00:10:00 at 100 °C .
10m
Neutralize the extract by adding 10 µL of 1M TRIS-HCl (pH8).
Vortex thoroughly, make sure tissue is at the bottom of the Eppendorf (spin down if needed).
Carefully transfer the lysate in a clean Eppendorf tube, do not carry over hair/tissue.
Sample can be stored at 4°C (short term) or at -20°C (long term).
Atp13a4(KO) genotyping
1h
Carry out the PCR process under the UV light cabinet. Prepare a Working Dilution (10 µM) of the PCR primers.
| A | B | C | D | |
| PCR primers | Sequence 5’ – 3’ | Stock [] | Working [] | |
| Atp(50715)CF | GGT CCC AGA ATT CCT TGG CA | 100 µM | 10 µM | |
| Atp(50715)WTR | CAG TGC TGA CAG GGA GAT CC | 100 µM | 10 µM | |
| Atp(50715)KOR | GCC TCA CAC TGC ACT CTT CT | 100 µM | 10 µM |
Prepare a mix for all samples containing the following components:
| A | B | C | |
| Component | Amount/Sample | Final conc | |
| Atp(50715)CF primer (10 µM) | 1 µl | 400 nM | |
| Atp(50715)WTR primer (10 µM) | 1 µl | 400 nM | |
| Atp(50715)KOR primer (10 µM) | 1 µl | 400 nM | |
| PCR mix (2x) | 12.5 µl | 1x | |
| H2O | 7.5 µl | -- |
Note
Prepare positive and negative controls per gene and prepare extra volume to account for pipetting variation.
To each PCR tube, add 23 µL from the reaction mixture and 2 µL gDNA (+/- 100ng) from each sample. Keep tubes On ice .
Run PCR protocol.
| A | B | C | D | |
| Initial denaturation | 95°C | 2’ | ||
| Denaturation | 95°C | 15” | X30 | |
| Annealing | 56°C | 15” | ||
| Elongation | 72°C | 30" | ||
| Final extension | 72°C | 7’ | ||
| Hold | 4°C | ¥ |
Prepare a 1.5% agarose gel in TBE buffer containing Midori Green (1:10,000).
Run the samples for 01:00:00 at 120V.
1h
Visualize the gel.
Expected result
| Results | Bands | Notation | |
| Wild type | 410 bp | WT | |
| Knock-out | 288 bp | KO |