Stock No: 012666
Protocol 348: Restriction Enzyme Digest Assay - Lepr<db>
Version 1.1

Notes

Publications are available which describe other approaches to genotyping this strain.  One of these can be found at: https://www.nature.com/articles/aps201752

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 = 108 bp and 27 bp
Heterozygote = 27 bp, 108 bp and 135 bp
Wild type = 135 bp

Male mice have an unknown artifact band of ~ 170bp.

Add DNA Loading Dye** to digested products and separate on a 3% agarose gel.***

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR0985 AGA ACG GAC ACT CTT TGA AGT CTC Forward
oIMR0986 CAT TCA AAC CAT AGT TTA GGT TTG TGT Reverse

Reaction A

Component Final Concentration
ddH2O
AB PCR BufferII 1.00 X
MgCl2 2.00 mM
dNTP 0.20 mM
oIMR0985 1.00 uM
oIMR0986 1.00 uM
Taq DNA Polymerase 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 52.0 --
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
RsaI

Strains Using This Protocol

Stock Number Strain Name
002048 B6 x C57BLKS-Dock7m Leprdb Myo15sh2-J/J
000697 B6.BKS(D)-Leprdb/J
000699 B6.Cg-Dock7m Leprdb/+ +/J
005089 B6.Cg-Qkqk-2J/GrsrJ
000642 BKS.Cg-Dock7m +/+ Leprdb/J
000700 BKS.Cg-Dock7m Leprdb/+ +/J
008340 BKS.Cg-Leprdb Nos3tm1Unc/RhrsJ
001192 BKS.Cg-meaJ Leprdb +/+ + Dock7m/J
000707 CBA.Cg-Dock7m Leprdb/+ +/J
012666 D2.BKS(D)-Leprdb/J
006654 FVB.BKS(D)-Leprdb/ChuaJ
11 strains use this protocol