Protocol 37257: Probe Assay - Yif1a<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

>chr19:5091599+5091696 98bp GAGGTGTTGGCTCTGCTTCT CCCACATATTTGTAACCGCTGTAAG

Mut = 110 bp

Wt = 98 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GAAATCAGGCTGGTACCTGTCTAGGATGAGCAGAGGTCTTGACCGTGGTGTTAATTCTGGAGATATTGCAGGGAGCTGATGAGTTTCCTAATGGGAGGGAAGGTGATGGCTATGGTCCTGGGAGGCCCAGTAGTAGTCAGCTGGAAGGGGCAGTCGAGAGGcgcaccggggacaggacattggagagtgaagttgggatatgcagggagggaaggatgggagaggctggagcactagtggacttgaatgcctaatgaggggctctgggtgaggaagccagaggaactacttcctctcttctcttctcttggcagccatggccttcatcacatacgtgctgctggctggaatggcgctaggcattcagcaaaggttagtattctcttctccctcctggcttatgtcccttcctctgtagggaatctgtgccaggtcctagctcacccaccttgggcctaccctgacctaccttctcccatggctgcaggttctccccagaggtgttgggcctgtgtgcaagcacagcactggtgtgggtgttcatggaggtgttggctctgcttctgggcctctacctggccaccgtacgcagtgaactgagtaccttccacctcttggcttacagcggttacaaatatgtggggtgagtgcctgcaggctgagaggagcctgaagggtctcaggctacaggttttgggcctatgtagcaacagccttcactgtgatgctagagacagttctactccgtctcaaccgccagctctacgctgtgagttggtggctgtttctgacagtctccccttggagCCAGTGACTTTAAAGAGACTGGGGATTTGTTAGTTACCTTGGGGAGCCCAGAGAAGGGAAGGGGAGTTTAGGCCCTGGGACTGAGTCCTGAACCTTGCAGGTCTATTTGGAGTCTGGTTCTTTGTA

This mutation is a 646 bp deletion beginning at Chromosome 19 position 5,091,215 bp and ending after 5,091,860 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50911 GAG GTG TTG GCT CTG CTT CT Wild type Forward A
50912 CCC ACA TAT TTG TAA CCG CTG TAA G Wild type Reverse A
50913 GGG AGG CCC AGT AGT AGT CA Mutant Forward A
50914 AAC TCC CCT TCC CTT CTC TG Mutant Reverse A
50915 Fluorophore-1 CAC CGT ACG CAG TGA ACT GAG Quencher-1 WT Probe
50916 Fluorophore-2 AGA GGC CAG TGA CTT TAA AGA GAC TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50911 0.40 uM
50912 0.40 uM
50913 0.40 uM
50914 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.