Protocol 30821: Probe Assay - Il17a<tm1Bcgen> 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

>chr1:20733850+20733951 102bp GGCAGCCTAAACAGAGACC GCTGAAAATCAATAGCACGAAC

Mutant=  106 bp

Wild Type = 102 bp

Sequence

Wt Sequence: GGCAGCCTAAACAGAGACCCGCGGCTgacccctaagaaacccccacgtttctcagcaaacttacttgcatttttaaaacagttcgtgctattgattttcagc

 

Mutant Sequence: GGCAGCCTAAACAGAGACCCGCGGCTagcagatctctcgaggttaacgaattccgccccccccccctaacgttactggccgaagccgcttggaataaggccggtgt

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
13007 ACA CCG GCC TTA TTC CAA G Mutant Reverse A
35590 GGC AGC CTA AAC AGA GAC C Common A
35591 GCT GAA AAT CAA TAG CAC GAA C Wild type Reverse A
35592 Fluorophore-1 AGC AGA TCT CTC GAG GTT AAC G Quencher-1 MUT Probe
35593 Fluorophore-2 CCC TAA GAA ACC CCC ACG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
13007 0.40 uM
35590 0.40 uM
35591 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.