Protocol 18858: QPCR Assay - Tg(SNCAcDNA)-qPCR
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 Sncatm1Rosl/J
004479 B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J
032799 C57BL/6-Tg(Thy1-SNCA*E35K*E46K*E61K)3798Nuber/J
017678 FVB;129-Pink1tm1Aub Tg(Prnp-SNCA*A53T)AAub/J
010710 FVB;129S6-Sncatm1Nbm Tg(SNCA)1Nbm/J
010788 FVB;129S6-Sncatm1Nbm Tg(SNCA*A30P)1Nbm Tg(SNCA*A30P)2Nbm/J
010799 FVB;129S6-Sncatm1Nbm Tg(SNCA*A53T)1Nbm Tg(SNCA*A53T)2Nbm/J
027344 STOCK Gbatm1Rlp Sncatm1Nbm Tg(SNCA)1Nbm/Mmjax
027227 STOCK Gbatm1Rlp Sncatm1Nbm Tg(SNCA*A53T)1Nbm Tg(SNCA*A53T)2Nbm/Mmjax
9 strains use this protocol