Protocol 30074 - Dock7<m>-Probe

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 = retrotransposon LTR insertion

Sequence

Wild Type Dock7

acataatgatttcaataacagacacgtgtcttttttatttaaagTGGATACCAATGCTGCAG

AATGGACGGTTGAAGACAGGGCAGTTCTGCCTGCCAGTCTCCCTGGAAAAACCACCGCAGGC

TTATTCAGTGCTTTCCCCCGAGgtcagtgccttccagcaag

Dock7<m>

ACATAATGATTTCAATAACAGACACGTGTCTTTTTTATTTAAAGTGGATACCAATGCTGCAG

AATGGACGGTTGAAGACCAGTTGCAGAAACGAGGATTATAAAGTAATATGAATGCCCATTGT

AAATTTACTAATGCGTTTGCGATTGTACGAGGGATAGTTAT

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction
14825		AGT GGA TAC CAA TGC TGC AG		Common	A
14826		TGG TTT TTC CAG GGA GAC TG		Wild type Reverse	A
14828		GGG CAT TCA TAT TAC TTT ATA ATC CTC G		Mutant Reverse	A
33838	Fluorophore-1	TCT GCA ACT GGT CTT CAA CCG TCC	Quencher-1	MUT Probe
33839	Fluorophore-2	ATG GAC GGT TGA AGA CAG GGC A	Quencher-2	WT Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
14825	0.40 uM
14826	0.40 uM
14828	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
000027	B6.D-Tyrp1^b Dock7^m/J
009659	B6.D2(BKS)-Dock7^m/J
000642	BKS.Cg-Dock7^m +/+ Lepr^db/J
031579	BKS.Cg-Dock7^m+/+ Lepr^db Tg(SLC2A4)HJdm/PfzrJ
001192	BKS.Cg-mea^J Lepr^db +/+ + Dock7^m/J
5 strains use this protocol