Protocol 22100: Probe Assay - Adrb1<tm1Bkk>-Probe
Version 2.0

Notes

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

Mutant=  225 bp

Wild Type = 113 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
15244 TTG GGA AGA CAA TAG CAG GC Mutant Forward A
19912 CGC TGT CCA CAG TGG TTG T Common A
22481 ATC TGG TCA TGG GAT TGC TG Wild type Forward A
22482 ACA CAG CAC ATC TAC CGA AG Wild type Reverse A
22483 Fluorophore-1 CTC GCA GAA GAA GGA GCC GTA CT Quencher-1 WT Probe
22485 Fluorophore-2 TTC CGC CTC AGA AGC CAT AGA GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
15244 0.40 uM
19912 0.40 uM
22481 0.40 uM
22482 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 4.0 -- Forever
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
031490 FVB.129-Adrb1tm1Bkk/J
003810 STOCK Adrb1tm1Bkk Adrb2tm1Bkk/J
2 strains use this protocol