Protocol 348: Restriction Enzyme Digest Assay - Lepr<db>

BKS.Cg-Dock7^m +/+ Lepr^db/J

Stock No: 000642

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-Dock7^m Lepr^db Myo15^sh2-J/J
000697	B6.BKS(D)-Lepr^db/J
000699	B6.Cg-Dock7^m Lepr^db/+ +/J
005089	B6.Cg-Qki^qk-2J/GrsrJ
000642	BKS.Cg-Dock7^m +/+ Lepr^db/J
000700	BKS.Cg-Dock7^m Lepr^db/+ +/J
008340	BKS.Cg-Lepr^db Nos3^tm1Unc/RhrsJ
001192	BKS.Cg-mea^J Lepr^db +/+ + Dock7^m/J
000707	CBA.Cg-Dock7^m Lepr^db/+ +/J
012666	D2.BKS(D)-Lepr^db/J
006654	FVB.BKS(D)-Lepr^db/ChuaJ
11 strains use this protocol