Protocol 18858: QPCR Assay - Tg(SNCAcDNAorGeniomic)-qPCR

B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J

Stock No: 004479

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

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
oIMR1544		CAC GTG GGC TCC AGC ATT		Internal Positive Control Forward	A
oIMR1770		TGA CGG GTG TGA CAG CAG TAG		Transgene	A
oIMR1771		CAG TGG CTG CTG CAA TG		Transgene	A
oIMR3580		TCA CCA GTC ATT TCT GCC TTT G		Internal Positive Control Reverse	A
TmoIMR0025	Fluorophore-1	CCC TGC TCC CTC CAC TGT CTT CTG G	Quencher-1	MUT Probe
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
oIMR1544	0.40 uM
oIMR1770	0.40 uM
oIMR1771	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
023837	B6.Cg-Tg(SNCA)OVX37Rwm Snca^tm1Rosl/J
004479	B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J
038895	B6;D2-Tg(Mbp-SNCA)1Ema/RoriJ
038896	B6;D2-Tg(Mbp-SNCA)29Ema/RoriJ
029124	C57BL/6-Gba1^tm1.1Mjff Tg(Thy1-SNCA)15Mjff/J
038021	C57BL/6-Tg(Th-SNCA*)MI2Mgsp Del(6Snca)1Slab/J
032799	C57BL/6-Tg(Thy1-SNCA*E35K*E46K*E61K)3798Nuber/J
018442	C57BL/6N-Tg(SNCA)129Mjff/J
017678	FVB;129-Pink1^tm1Aub Tg(Prnp-SNCA*A53T)AAub/J
010710	FVB;129S6-Snca^tm1Nbm Tg(SNCA)1Nbm/J
010788	FVB;129S6-Snca^tm1Nbm Tg(SNCA*A30P)1Nbm Tg(SNCA*A30P)2Nbm/J
010799	FVB;129S6-Tg(SNCA*A53T)1Nbm Snca^tm1Nbm Tg(SNCA*A53T)2Nbm/J
027227	STOCK Gba1^tm1Rlp Tg(SNCA*A53T)1Nbm Snca^tm1Nbm Tg(SNCA*A53T)2Nbm/Mmjax
037870	STOCK Snca^tm1Rosl Tg(Cnp-SNCA)M2Vle/KlukJ
14 strains use this protocol