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Genotype analysis of knockout mice

MR Matthew J. Robertson
KK Katarzyna Kent
NT Nathan Tharp
KN Kaori Nozawa
LD Laura Dean
MM Michelle Mathew
SG Sandra L. Grimm
ZY Zhifeng Yu
CL Christine Légaré
YF Yoshitaka Fujihara
MI Masahito Ikawa
RS Robert Sullivan
CC Cristian Coarfa
MM Martin M. Matzuk
TG Thomas X. Garcia
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For Sanger sequence analysis of mutant mice, genomic DNA was isolated by incubating tail tips in lysis buffer [20 mM Tris-HCl (pH 8.0), 5 mM EDTA, 400 mM NaCl, 0.3% SDS, and 200 μg/mL Actinase E solution] at 60 °C overnight. Polymerase chain reactions (PCRs) amplifying the genomic region containing the insertion/deletion events were performed using KOD Xtreme enzyme (TOYOBO, Osaka, Japan); PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Carlsbad, CA, USA) and sent for Sanger sequencing on an ABI 3130XL Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) using the forward primer. For routine genotyping of mutant mice, genomic DNA was isolated by separately incubating ear snips and tail tips in 50 mM NaOH solution at 95 °C overnight and inactivating with 1 M Tris pH = 8.0. PCRs amplifying wild-type and mutant-specific amplicons were performed using 2X amfiSure PCR Master Mix (GenDEPOT, Barker, TX). Primer sequences are listed in Additional file 1: Table S13.

Copyright and License information: The Author(s) ©2020
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