Protocol 22184: QPCR Assay - Generic Hygromycin

B6.129P2-Pzp^tm1Vln Mug1^tm2Vln/J

Stock No: 024839

Version 7.0

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	Note
13585	Fluorophore-1	AAG ACC TGC CTG AAA CCG AAC TGC C	Quencher-1	Tg Probe
oIMR1219		CGA GAG CCT GAC CTA TTG CAT		Transgene	A
oIMR1220		CGA CCG GCT GCA GAA CA		Transgene	A
oIMR1544		CAC GTG GGC TCC AGC ATT		Internal Positive Control Forward	A
oIMR3580		TCA CCA GTC ATT TCT GCC TTT G		Internal Positive Control Reverse	A
TmoIMR0105	Fluorophore-2	CCA ATG GTC GGG CAC TGC TCA A	Quencher-2	IC Probe

Reaction A

Component	Final Concentration
ddH2O
Kapa Probe Fast QPCR	1.00 X
oIMR1219	0.40 uM
oIMR1220	0.40 uM
oIMR1544	0.40 uM
oIMR3580	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

Stock Number	Strain Name
024839	B6.129P2-Pzp^tm1Vln Mug1^tm2Vln/J
035404	B6;129S-Igs7^tm166(tetO-EGFP*,CAG-tTA2)Hze/XwyJ
034797	B6;129S4-Gt(ROSA)26Sor^tm2(CAG-rtTA3,-mKate2)Slowe Col1a1^tm1(tetO-GFP,-PRKAA1*)Rjsh/J
034422	B6;129S6-Slc17a7^em1(flpo)Tasic/J
014594	C;129S4-Rag2^tm1.1Flv Thpo^tm1.1(TPO)Flv Il2rg^tm1.1Flv/J
003127	FVB;129P2-Bcl3^tm1Ver/J
035561	STOCK Tc(HSA21,CAG-EGFP)1Yakaz/J
7 strains use this protocol