Stock No: 028104
Protocol 21646: Probe Assay - Ebf1<tm2.1Mbu> Probe
Version 1.0

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=  106 bp

Wild Type = 96 bp

Sequence

Wt Sequence: CAAGAAAAGCTGTGGCAACCGAAATGAGacTCCCTCAGATCCAGTGATAATTGACAGgtagggaccactcttctttcactggcgtcttagtcctca

 

Mutant Sequence: CAAGAAAAGCTGTGGCAAccGAAATGAGACTCCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAAC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
24626 GTT TAC GTC GCC GTC CAG Mutant Reverse A
32921 CAA GAA AAG CTG TGG CAA CC Common A
32922 TGA GGA CTA AGA CGC CAG TG Wild type Reverse A
32923 Fluorophore-1 AGG AGC TGT TCA CCG GG Quencher-1 MUT Probe
32924 Fluorophore-2 TGA CAG GTA GGG ACC ACT CTT C Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
24626 0.40 uM
32921 0.40 uM
32922 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 40.0 -- Forever
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.