Genotyping protocol for mice
vanessa promes
Published: 2023-06-27 DOI: 10.17504/protocols.io.kxygx3mjwg8j/v1
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Abstract
This protocol describes the general method to genotype the mice.
Steps
DNA Extraction
1.
Obtain tail sample from mice.
2.
Add 100µL of NaOH into tail sample and place in hot plate at 100C for 0h 10m 0s. Vortex sample and place in hot plate once more for 0h 10m 0s.
3.
Add 50µL of 1M Tris pH 8.0 to neutralize extraction
4.
Spin for 20,000xg for 0h 10m 0s
5.
Use 2µL in PCR reaction.
Preparing PCR reaction
6.
Obtain a 1.5ml microcentrifuge tube and add 10ul 2x DreamTaq Green Master mix. 2ul forward and reverse primers, 2µL DNA lysate and Nuclease-free water up to 20µL
7.
Thermocycling parameters:
95oC 5min
95oC 30sec| x 30 cycles
60oC 30sec|
72oC 1min|
72oC 2min
4oC hold
Run DNA Sample
8.
In 2% agarose in TAE buffer add 10ul of the sample and run for around 30min
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