Protocol 30006: QPCR Assay - SMN Genomic

FVB.Cg-Grm7^{Tg(SMN2)89Ahmb} Smn1^tm1Msd/2J

Stock No: 007949

Version 5.0

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

Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
13743	Fluorophore-1	CTG TCT CCA GAT AAT TCC CCC ACC ACC T	Quencher-1	MUT Probe
13745	Fluorophore-2	CCA ATG GTC GGG CAC TGC TCA A	Quencher-2	MUT Probe
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
oIMR4139		AAT CTC CAG ACT TTA CTT TTT TGT TTA CTG		Mutant Forward	A
oIMR4140		TCA TCA AGA GAA TCT GGA CAT ATG G		Mutant Reverse	A

Reaction A

Component	Final Concentration
ddH2O
Kapa Probe Fast QPCR	1.00 X
oIMR1544	0.40 uM
oIMR3580	0.40 uM
oIMR4139	0.40 uM
oIMR4140	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
008631	B6.Cg-Tg(SMN2)11Tro Tg(SMN2)46Tro Smn1^tm1Msd/J
005025	FVB.Cg-Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(SMN2*delta7)4299Ahmb/J
007949	FVB.Cg-Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd/2J
008783	STOCK Grm7^Tg(SMN2)89Ahmb Smn1^tm3(SMN2/Smn1)Mrph Tg(CAG-cre/Esr1*)5Amc Tg(SMN2*delta7)4299Ahmb/J
4 strains use this protocol