Jul 02, 2024

Public workspaceFast rodent genotyping

Science Advances
DOI
dx.doi.org/10.17504/protocols.io.bp2l627k5gqe/v1
  • 1Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065, USA;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland 20815, USA
Alexandros C Kokotos
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DOI: dx.doi.org/10.17504/protocols.io.bp2l627k5gqe/v1
Protocol Citation: Alexandros C Kokotos, Timothy A. Ryan 2024. Fast rodent genotyping. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l627k5gqe/v1
Manuscript citation:
Phosphoglycerate kinase is a central leverage point in Parkinson’s Disease driven neuronal metabolic deficits
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: June 27, 2024
Last Modified: July 02, 2024
Protocol Integer ID: 102548
Keywords: ASAPCRN, genotyping, sexing, PCR, tail clip, genotype
Funders Acknowledgements:
ASAP
Grant ID: 000580
Abstract
This protocol describes a very fast method (approximately 2 h) to do genotyping from rodent tail clips and other samples.
Materials
Equipment
Operating Scissors (like Roboz Surgical Store RS-6828)
70% Ethanol mix (KOPTEC UN1170)
Heat block (like Thermo Fisher 88870002)
PCR thermocycler (like Bio-Rad MyCycler)
DNA electrophoresis system (like VWR Horizontal MINI M Gel Electrophorosis System)
DNA gel visualization device (like Licor Odyssey Fc Imager)
Mini centrifuge (like USA Scientific 2631-0006)
Microwave oven

Consumables
Platinum Direct PCR Universal Master Mix (Sigma Aldrich A44647100)
Eppendorf tubes (labForce 1149K01)
PCR tubes (Bio-Rad TWI0201)
Tris (Sigma Aldrich T6066)
EDTA (Sigma Aldrich E6511)
Glacial acetic acid (Sigma Aldrich AX0073)
Agarose (Thermo Fisher 16500-500)
DNA dye (Biotium 41011)
DNA ladder (like NEB N0550 or N0556)

Recipes
50x TAE buffer, 1L in ddH2O
2 M Tris (242 g)
1 M Glacial acetic acid (60.05 mL)
50 mM EDTA (20.81 g)




Before start
The current protocol describes genotyping using rodent tail clips. This protocol has been verified using both rats (Sprague Dawley strain) and mice (C57BL/6J strain). It can also be applied to other types of biological samples, like blood, buccal swabs etc.
Example genotyping applied includes PARK20 mice and sexing of wt rats.
Operating scissors should be kept very sharp at all times, by cutting through aluminum foil or stainless steel sponges.
DNA extraction
DNA extraction
10m
10m
Tail clips from mice or rats of appropriate age should be snipped using sharp operating scissors and in compliance to local protocols by national and institutional regulatory organizations and placed in sterile Eppendorf tubes.
Note: This protocol has been tested using the smallest tail clips possible of ~ 1 mm. Always clean the scissors with Concentration70 % (v/v) Ethanol in between animals.
Add Amount20 µL of Lysis Buffer and Amount0.6 µL of Proteinase K in each sample tube.
Note: Preparing a master mix is suggested for several samples.
Incubate samples at TemperatureRoom temperature for Duration00:01:00 .
1m
Put samples in the heat block at Temperature98 °C for Duration00:01:00 .

1m
Spin down the samples using a mini-centrifuge for Duration00:00:30 and transfer lysate to new tubes.

30s
PCR
PCR
1h 20m
1h 20m
Prepare PCR reactions at TemperatureRoom temperature as follows
Note: Preparing a master mix is suggested for several samples.
AB
ComponentVolume (ul)
Platinum master mix10
DNA sample1
Primers0.4+0.4
Nuclease free water8.2
Total20



Note: PCR reaction may need optimization. Primers should be used at a final concentration of Concentration0.2 micromolar (µM) . In this example, Amount0.4 µL is added for each of two primers from a Concentration10 micromolar (µM) stock.

Place PCR samples in a PCR thermocycler. Set up the thermocycler with the following program using a Hot Start function.
ABCD
StepTemperature (°C)TimeCycles
Initial Denaturation942 min1
Denaturation9415 s35
Annealing5515 s35
Extension6820 s/Kb35
Final Extension682 min1
Hold4oo1

Note: PCR program may need optimization.
DNA gel electrophoresis
DNA gel electrophoresis
30m
30m
During the PCR, prepare an appropriate agarose percentage DNA gel.
Load PCR sampes along with an appropriate DNA ladder on the DNA gel.
Submit the DNA gel to electrophoresis.
Visualize gel using an appropriate device.
Protocol references
M. Cao et al., Parkinson Sac Domain Mutation in Synaptojanin 1 Impairs Clathrin Uncoating at Synapses and Triggers Dystrophic Changes in Dopaminergic Axons. Neuron 93, 882-+ (2017).

P. Dhakal, M. J. Soares, Single-step PCR-based genetic sex determination of rat tissues and cells. Biotechniques 62, 232-233 (2017).