Sex genotyping of mice (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Martin Cohn

Keywords

[‘mouse’]

Subjects

[‘Developmental biology’, ‘Isolation, purification and separation’, ‘Molecular biology’]

Release Date

2017-08-07

Abstract

Sex genotyping of mice protocol by the Cohn Group.

Procedure

The sex genotyping method used by the Cohn lab is based on the highly homologous SMC genes found on the X (X chromosomal SmcX = Kdm5c) and Y (Y chromosomal SmcY = Kdm5d) chromosomes.

Homologous pair SmcX/SmcY

• SMCX-1 5’-CCGCTGCCAAATTCTTTGG-3’

• SMC4-1 5’-TGAAGCTTTTGGCTTTGAG-3’

• PCR product: females a single band (350 bp), males 2 bands (350 & 300 bp) because of an intron difference between the X and Y genes (Agulnik 1997, PMID 9060413) adapted from Case Western Transgenic Facility.

DNA extraction

1. Add tissue, forelimb of embryo,** **to 300µl of 25mM NaOH.

2. Heat at 95ºC for 2 hours.

3. Add 300µl 40mM Tris HCl.

4. Store at 4ºC till genotyping.

PCR mix

1. Set up the PCR mix as follows:

   16.25µl H20

   5µl 5X Green GoTaq Reaction Buffer (Promega cat #M791B)

   1µl Primer X (CCGCTGCCAAATTCTTTGG, Integrated DNA Technologies)

   1µl Primer Y (TGAAGCTTTTGGCTTTGAG, Integrated DNA Technologies)

   0.5µl dNTP mix ~ 10mM (Thermo Scientific cat #R0192)

   0.25µl Taq DNA Polymerase with ThermoPol Buffer (New England BioLabs cat #M0267L)

   1µl DNA

PCR Cycling Conditions

1. 94ºC 3:00 minutes 1 cycle

2. 94ºC 30 seconds 35 cycles

   54ºC 30 seconds

   72ºC 30 seconds

3. 72ºC 7:00 minutes 1 cycle

4. 10ºC hold

Analysis of the PCR products on an agarose gel

1. 5µl of PCR product is run on a 2% agarose gel.

2. Female mice are identified by a single 350bp band and males are identified by 2 smaller bands of 300bp & 350bp.

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium