May 20, 2026
Atp13a2 Flox and Atp13a2 Null genotyping
- 1KU Leuven
Protocol Citation: Hanne Dhondt, Ana Cascalho, Aisha Sati 2026. Atp13a2 Flox and Atp13a2 Null genotyping. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9jnxv4o/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: March 05, 2025
Last Modified: May 20, 2026
Protocol Integer ID: 123864
Keywords: ASAPCRN, protocol details the atp13a2 flox, atp13a2 flox, null genotyping, flox, protocol detail, null
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-024297
Abstract
This protocol details the Atp13a2 Flox and Atp13a2 Null genotyping.
Materials
- gDNA isolated from ear/tail biopsies:
- Genotyping mix: PCR Bio Rapid 2x (PCR bio, PB10.23)
- PCR tubes
- UltraPure™ Agarose
- Midori Green Nucleic Acid Staining Solution (Bulldog-bio; cat no MG04)
- DNA Ladder (Low range DNA ladder, 100 − 1000 bpVWR International (Avantor)Catalog #733-2602 OR
SmartLadder - 200 to 10000 bpEurogentecCatalog #MW-1700-10 )
PCR primers:
| A | B | C | D | |
| PCR primers | Sequence 5’ – 3’ | Stock | Working | |
| Forward primer | CTG CAG CTT CGA GAG GAA AG | 100 µM | 25 µM | |
| Reverse primer (WT/Flox) | CAC TCT GTC CTC AGG CCT TC | 100 µM | 25 µM | |
| Reverse primer 1 (KO) | TGA GAA GTG GGA ATC GGG | 100 µM | 25 µM |
PCR mix:
| A | B | C | |
| Component | Amount | Total concentration | |
| F primer (25 µM) | 0.75 µl | 750 nM | |
| R primer (25 µM) | 0.75 µl | ||
| R primer 1 (25 µM) | 0.75 µl | ||
| PCR mix (2x) | 12.5 µl | ||
| H2O | 8.75 µl | ||
| DNA | 1.5 µl | ||
| Total volume | 25 µl |
Double gel :
| A | B | |
| TBE | 250 ml | |
| Agarose powder | 3.75 g | |
| Midori green | 25 µl |
Single gel:
| A | B | |
| TBE | 150 ml | |
| Agarose powder | 2.25 g | |
| Midori green | 15 µl |
Safety warnings
Carefully handle the midori green and correctly dispose it in a separate waste container.
Atp13a2 Flox and Atp13a2 Null genotyping
Carry out the PCR process under the UV light cabinet. Prepare a Working Dilution (25 micromolar (µM) ) of the PCR primers.
| A | B | C | D | |
| PCR primers | Sequence 5' - 3' | Stock | Working | |
| Forward primer | CTG CAG CTT CGA GAG GAA AG | 100 µM | 25 µM | |
| Reverse primer (WT/Flox) | CAC TCT GTC CTC AGG CCT TC | 100 µM | 25 µM | |
| Reverse primer 1 (KO) | TGA GAA GTG GGA ATC GGG | 100 µM | 25 µM |
Prepare a mix for all samples containing the following components:
Note
Prepare positive and negative controls per gene and prepare extra volume to account for pipetting variation.
| A | B | C | |
| Component | Amount | Final conce | |
| F primer (25 µM) | 0.75 µl | 750 nM | |
| R primer (25 µM) | 0.75 µl | 750 nM | |
| R primer 1 (25 µM) | 0.75 µl | 750 nM | |
| PCR mix (2x) | 12.5 µl | 1x | |
| H2O | 8.75 µl | -- | |
| DNA | 1.5 µl | ||
| Total volume | 25 µl |
To each PCR tube, add 1.5 µL gDNA from each sample and 23.5 µL from the reaction mixture.
Run PCR protocol
| A | B | C | D | |
| Initial denaturation | 94 °C | 3' | ||
| Denaturation | 94 °C | 30'' | X30 | |
| Annealing | 58 °C | 30" | ||
| Elongation | 72 °C | 30" | ||
| Final extension | 72°C | 10' | ||
| Hold | 4 °C | |
Prepare a 1.5% agarose gel in TBE buffer containing Midori Green (1:10000).
- Double gel:
| A | B | |
| TBE | 250 ml | |
| Agarose powder | 3.75 g | |
| Midori Green | 25 µl |
- Single gel:
| A | B | |
| TBE | 150 ml | |
| Agarose powder | 2.25 g | |
| Midori Green | 15 µl |
Dilute TBE from the stock (10x) to a 1x in distilled water.
Weight and add the agarose powder in a dedicated glass bottle together with TBE.
Mix them together.
Heat the solution in the microwave.
Cool it down by swirling underneath cold water.
Add the Midori Green nucleic acid staining solution and mix it again.
Pour it in the gel system and remove the bubbles (using a tip). Insert the combs and wait for its gelification (+/- 20 min).
Place the gel in the system and cover with TBE buffer.
Vortex ladder prior to loading to ensure even glycerol distribution. Load the samples (25 µL ) + DNA ladder (15 µL ) into one well of the gel .
Note
Do not leave more than 4 wells without any sample to avoid the smiling effect.
Run the samples for 01:20:00 at 120 V.
Note
Black is negative, red is positive. (The DNA is negatively charged and will run towards the positive electrode.) Always run to red!
1h 20m
Visualize the gel in the machine Gene Flash (Syngene Bio Imaging; Westburg, VWR) OR via Bio-Rad Chemidoc MP Imaging System.
| A | B | C | |
| Results | Band | Notation | |
| Floxed | 564bp | Flox | |
| Wild type | 426bp | WT | |
| Knock-out | 256bp | KO |