Protocol 22119 - Ddr2<slie>/JngJ

Notes

Taqman qPCR protocols are run on an ABI 7500, 7700, 7900 or the Roche LightCycler
480. 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

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction
oIMR1544		CAC GTG GGC TCC AGC ATT		Internal Positive Control	A
oIMR3580		TCA CCA GTC ATT TCT GCC TTT G		Internal Positive Control Reverse	A
oIMR9641		GAG AGA TGG CTC AAT CAT TAA AGG		Transgene	A
oIMR9642		AGC TGT GGG TTA GAA GAT AAG ATC C		Transgene	A
TmoIMR0105	Fluorophore-1	CCA ATG GTC GGG CAC TGC TCA A	Quencher-1	IC Probe
TmoIMR0143	Fluorophore-2	CAA CCA AAA TTA AGA CAA AAA CTC AAG GCA	Quencher-2	MUT Probe

Reaction A

Component	Final Concentration
ddH2O
Kapa Probe Fast QPCR	1.00 X
oIMR1544	0.40 uM
oIMR3580	0.40 uM
oIMR9641	0.40 uM
oIMR9642	0.40 uM
Tg Probe	0.15 uM
IC 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

This is the only strain that uses this protocol.