Stock No: 002019
Protocol 170: Restriction Enzyme Digest Assay - Foxn1<nu>
Version 1.1

Notes

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Mutant = ~110 bp and 58 bp
Heterozygote = 20 bp, 53 bp, 58 bp, 90 bp and ~110 bp
Wild type = 90 bp, 53 bp and 20 bp
Separate digested PCR product on a 5% agarose gel.**

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR1292 GGC CCA GCA GGC AGC CCA AG Common
oIMR1293 AGG GAT CTC CTC AAA GGC TTC Common

Reaction A

Component Final Concentration
ddH2O
AB PCR BufferII 1.00 X
MgCl2 2.50 mM
dNTP 0.20 mM
oIMR1292 1.00 uM
oIMR1293 1.00 uM
DNA Loading Dye 0.69 mM
Taq DNA Polymerase 0.02 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 55.0 --
4 72.0 -- repeat steps 2-4 for 35 cycles
5 72.0 --
6 10.0 -- hold**
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Reaction B

Component Final Concentration
AB PCR BufferII X
ddH2O
BseDI

Strains Using This Protocol

Stock Number Strain Name
000819 B6.Cg-Foxn1nu/J
000711 CByJ.Cg-Foxn1nu/J
007850 J:NU
002019 NU/J
003118 STOCK Ces1ce Foxn1nu/J
5 strains use this protocol