Protocol 33866: Probe Assay - Tmie<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

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

>chr9:110870914-110870998 85bp CACTTGCTTCTTTGAGCCTTG GATCAGAGTCCCAGACCGTGT

Mut= 92 bp

Wt= 85 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 ACCCATGTAGTCCAGGCGTAGGGCTCTATTGGGAGACGAGAGCAAAACCAGTCTACCAGGCAGCACACCTGGCTCATGAAACAGGCCTTGCCTTGTCTCTGGCCTTGGTCCTGAGACCCCATCCGCGCTGACTAGGACAGACTGTCCACACTTGCTTCTTTGAGCCTTGTTTCCCCAAGCATCAACCACCcttcgagtccaagtccctggtgacacggtctgggactctgatcagtgctcttattctgaatcttggatttttttttgaggtctagggtgttggttgtgaatgtctgaaaccttttggggattctaaaatgtgtgggctgagtctttgctgaccccatcccagcctgctagcctgggcctggctcactctgttctctcttgacccacagcccagcaccgcaccgccaaagcccaagccgcccccactgaccaaggagactgtggtgttctgggacatgcgcctgtggcacgtggtgggcatcttttcgctcttcgtgttgtccatcagtgagtagctgtgccctgccctctccaccccaccacaggaggaacacagcccttctagcatccctggcccctaccgtatgccttgggttcagagctgcaattctgggcagttcaaataggctaagcacgcacaagaggcccaccgtggtctctggcacctcctcagtcacacaaagtccctgtggagggctgacgggccagcggtgacccaaagatttctgctgggcccagggaagcagccacatggcaattatgttaattataggggactccaactgaatcCAGGACATGTCATCTTTCCTATGGCTGGCTTCCTGATGCTTTTCTTGCACTTTGGCCAATTCAGGGAGCCCACACCCAGGGGCCCCAGCAAGGCCAGCCTCCACCAGAGGCTCCCCAGACGCCGAGCAGAGGCGGCTTCCAGACCCTGAGCCCTACCTCTTCAACTGCCATTTTC

This mutation is a 608 bp deletion beginning at Chromosome 9 position 110,870,349 bp and ending after 110,870,956 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42365 CAC TTG CTT CTT TGA GCC TTG Common A
42366 GAT CAG AGT CCC AGA CCG TGT Wild type Reverse A
42367 GTG CAA GAA AAG CAT CAG GA Mutant Reverse A
42368 Fluorophore-1 TCG AGT CCA AGT CCC TGG T Quencher-1 WT Probe
42369 Fluorophore-2 ACC ACC CAG GAC ATG TCA TC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42365 0.40 uM
42366 0.40 uM
42367 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.