Stock No: 001428
Protocol 17685: QPCR Assay - Mbp<shi>
Version 6.0

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.

Oligo 14576 replaces TmoIMR0082

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

The Target  probe is for WILD TYPE.  Therefore, Homs do NOT amplify for target. 

Wild Type will appear below hets on the graph.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
14576 Fluorophore-1 CAT CCG TCT TGC ATA ATG CTT GCT GTG A Quencher-1 Tg Probe 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
oIMR6166 GTG GCT GGG TTG CTT CTC A Transgene Forward A
oIMR6167 CCC TCA ATG TTC CGT GCT G Transgene Reverse A
TmoIMR0105 Fluorophore-2 CCA ATG GTC GGG CAC TGC TCA A Quencher-2 IC Probe A

Reaction A

Component Final Concentration
ddH2O
Kapa Probe Fast QPCR 1.00 X
14576 0.40 uM
oIMR1544 0.40 uM
oIMR3580 0.40 uM
oIMR6166 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

This is the only strain that uses this protocol.