Protocol 30559: Probe Assay - Taf13<em1(IMPC)J> 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

>chr3:108380912+108381034 123bp TGCCCAGCTATAATTGTGTTTG ACTGCGAGAGAGCAAGAGTG

Mutant=  119 bp

Wild Type = 123 bp

Sequence

Wt Sequence: tgcccagctataattgtgtttgaaccattgtttctaattgCGtttaatcctagttggtatggaaagtatactggtggcattggtcagtctctgagtcaggcaacactcttgctctctcgcagT

 

Mutant Sequence: tgcccagctataattgtgtttgaaccattgtttctaattgCTttgtgcaagttcctggtttctatttgccattactctgaatatcacatcaatgtatcccagtgcttcagagcttgttt

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34892 TGC CCA GCT ATA ATT GTG TTT G Common A
34893 ACT GCG AGA GAG CAA GAG TG Wild type Reverse A
34894 AAA CAA GCT CTG AAG CAC TGG Mutant Reverse A
34895 Fluorophore-1 TGG TGG CAT TGG TCA GTC T Quencher-1 WT Probe
34896 Fluorophore-2 TTG CCA TTA CTC TGA ATA TCA CAT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34892 0.40 uM
34893 0.40 uM
34894 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.