Protocol 31185 - Dock7<m> qPCR

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

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
14829	Fluorophore-1	TCT GCA ACT GGT CTT CAA CCG TCC	Quencher-1	MUT Probe
14848	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	--	repeat steps 2-3 for 40 cycles

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
009659	B6.D2(BKS)-Dock7^m/J
000642	BKS.Cg-Dock7^m +/+ Lepr^db/J
001192	BKS.Cg-mea^J Lepr^db +/+ + Dock7^m/J
3 strains use this protocol