Protocol 31940: QPCR Assay - Generic LacZ QPCR Alternate 1
Version 8.0


Taqman qPCR protocols are run on a real time PCR instrument. 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
37301 GTT TAT GCA GCA ACG AGA CG Transgene Reverse A
37302 Fluorophore-1 CAG GAT ATG TGG CGG ATG AGC Quencher-1 Tg Probe
mom0095 TGA CGG CAG TTA TCT GGA AG Transgene Forward A
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
37301 0.40 uM
mom0095 0.40 uM
oIMR1544 0.40 uM
oIMR3580 0.40 uM
Tg Probe 0.15 uM
IC Probe 0.15 uM


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
028635 B6.129(Cg)-Cntnap2tm2Pele/J
013144 B6.129P2-Aim2Gt(CSG445)Byg/J
034597 B6.129P2-Kif13aGt(AF0553)Wtsi/AhcJ
025101 B6.Cg-Avpr1atm1Dgen Tg(AVPR1A)1Bux/BuxJ
005317 B6.Cg-Tg(BAT-lacZ)3Picc/J
002982 B6.Cg-Tg(CAG-lacZ)32And/J
033345 B6.Cg-Tg(Cdh5-COP4*L132C,-lacZ)B28-2Mik/J
032889 B6.Cg-Tg(Cdh5-RHO/Adrb2,-lacZ)B27-3Mik/J
024050 B6.Cg-Tg(Hmgcr-lacZ)H253Sest/J
007972 B6;CBA-Tg(Olfr151-taulacZ)4Mom/MomJ
007973 B6;CBA-Tg(Olfr16-taulacZ)1Mom/MomJ
007974 B6;CBA-Tg(Olfr160-taulacZ)V4-7Mom/MomJ
007976 B6;CBA-Tg(Olfr713-taulacZ)4Mom/MomJ
007975 B6;CBA-Tg(OR8A1-taulacZ)1Mom/MomJ
008344 B6;DBA-Tg(Fos-tTA,Fos-EGFP*)1Mmay Tg(tetO-lacZ,tTA*)1Mmay/J
026216 B6;SJL-Tg(Tnnt2-lacZ)497Jl/J
034731 B6N(129S4)-Srd5a3tm1c(EUCOMM)Wtsi/J
035185 STOCK Adgrl1tm2c(EUCOMM)Hmgu/SudJ
007922 STOCK Gli2tm2.1Alj/J
005707 STOCK Rag1tm1Mom Tg(TIE2-lacZ)182Sato/J
027315 STOCK Vegfatm2.1Nagy/J
004623 STOCK Tg(TCF/Lef1-lacZ)34Efu/J
005728 STOCK Tg(tetO-Ipf1,lacZ)958.1Macd/J
23 strains use this protocol