Protocol 13582: QPCR Assay - Trisomy QPCR
Version 3.0

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
oIMR1863 TGC TGA AGA TGT GGG TTC GA
oIMR1864 GAC AAT CAC GGT TGC TAT GAC AA
oIMR3580 TCA CCA GTC ATT TCT GCC TTT G Internal Positive Control Reverse
TmoIMR0032 Fluorophore-1 CAA AGG CGC CAT CAT CGG ACT CA Quencher-1
TmoIMR0107 Fluorophore-2 CCA ATG GTC GGG CAC TGC TCA A Quencher-2 IC Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.91
ddH2O
oIMR1863 0.43
oIMR1864 0.43
oIMR1544 0.00
oIMR3580 0.00
TmoIMR0032 1.27
TmoIMR0107 0.00
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
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
001924 B6EiC3Sn a/A-Ts(1716)65Dn/J
004850 B6EiC3Sn-Rb(12.Ts171665Dn)2Cje/CjeDnJ
2 strains use this protocol