Protocol 21995: End Point Analysis Assay - Lepr<db>

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

Stock No: 000700

Version 7.0

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

Mut=T

Wt=G

Sequence

AGAACGGACACTCTTTGAAGTCTCTCATGACCACTACAGATGAACCCAATCTACC

AACTTCCCAACAGTCCATACAATATTAGAAGATGTTTACATTTTGATGGAGG(g/t)AA

ACAAACCTAAACTATGGTTTGAATGACTAAGAAATAACATTTGATGAGCTTATTAG

AGAAGTGTATATTTTGTGGCCACAATGTAGGTTTGATGTAGTTCAGTTTGGACATAT

GCTTGATTTTCAGGGCATCAAAAATTTA

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
14349		ACC AAC TTC CCA ACA GTC CA		Forward	A
14350		TGA TGC CCT GAA AAT CAA GC		Reverse	A
17934	Fluorophore-1	TTT GAT GGA GGG AAA CAA ACC TA	Quencher-1	WT Probe
17935	Fluorophore-2	TTT TGA TGG AGG TAA ACA AAC CTA A	Quencher-2	MUT Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
14349	0.40 uM
14350	0.40 uM
Wt Probe	0.15 uM
Mutant Probe	0.15 uM
DNA

Cycling

Step	Temp °C	Time	Note
1	95.0	--
2	95.0	--
3	60.0	--
4		--	repeat steps 2-3 for 40 cycles
5	40.0	--	Forever

JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

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
000642	BKS.Cg-Dock7^m +/+ Lepr^db/J
000700	BKS.Cg-Dock7^m Lepr^db/+ +/J
031579	BKS.Cg-Dock7^m+/+ Lepr^db Tg(SLC2A4)HJdm/PfzrJ
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