Protocol 22149: QPCR Assay - Tg(LRRK2)
Version 4.0

Notes

Amplifies an 84 bp product from Homo sapien leucine-rich repeat kinase 2. LMV 06/14/10
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
10649 GCA TTA GAG ATG TTA TCC CTG GAA Transgene Forward A
10650 GTA CTG ACC TTG GTC ATC TGG ATA Transgene Reverse A
10651 Fluorophore-1 ATG GAT TCA GTG CTT CAC ACA CTG CA Quencher-1 Tg 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
TmoIMR0105 Fluorophore-2 CCA ATG GTC GGG CAC TGC TCA A Quencher-2 IC Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
10649 0.40 uM
10650 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
018785 C57BL/6J-Tg(LRRK2*G2019S)2AMjff/J
018786 C57BL/6J-Tg(LRRK2*R1441G)3IMjff/J
009610 FVB/N-Tg(LRRK2)1Cjli/J
009609 FVB/N-Tg(LRRK2*G2019S)1Cjli/J
4 strains use this protocol