Protocol 37029: Probe Assay - Klhl34<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

>chrX:157818305+157818392 88bp AAGCTGAGCAGGCAAAAATC GCTGGAGGCATCCCATAAC

Mut = 118 bp

Wt = 88 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

AGCCAAATCTGGATGAAGGAACCTTAGGTTTTAAGCTGAGCAGGCAAAAATCAGCCTTTGCTAGGGGCTCAGCAGCTGgacagcggaggtgggggttccgtgttatgggatgcctccagccgcgctgggctggctccatcccagcttcccgccttgggcatcatgagttacttcctgtcttactgcaaaacccacggcagctctcttctcactggctaccaggccctacgcgctgagggcttcttgtgcgacgtgacactggaggccgagggcagtgagttcccagcacacaggtcacttctggcgtgctccagcgactacttcagggccctgttcaagagtcacactcaggaatcgcgggctcgcgtaattcacctgcgtgtgccttcagcggccggcctacagcgcctgctggacttcatctacactgcctggctgcccctctccatggacactgtggaggacactctggaggctgccagctacctgcaagtcaccgaggccttgggactgtgtgcccactacttggaacgccagcttgccccagagaattgctgtttcattgccaatgtggcagctcgatttggcctggcccgcaccctaggtgcagcggagtgctgtattgtacgccacctggggggcctgttactgatgggcactggcccctcggggctgttggaactcaatcctatatcaatgaaagctgtgctgggcgcccctgacgtggcacgggtgcctgagacgcggctgctgggcctggcactagcctggctgcagcaggaacccaaggtccagcgcctggcacatagttcaacactgcttggatgcatccgctttggcctggtgcctgctggtgtgctgcggcgcatatactcgggctccggtcttatattacctactaggatcaaaagcctcatcatccaggccctcaattaccatacagcaccctccagccagccgcttctgcagggagaacagactagtgttcggagtccccagacccgtatcttgctagttggtggtctcagagctagggagatggttaccgaggagatcatgggcctaccagaggtagttcagaatcggcaccctgcacctcaaccagaggaagaggagggacaggaggaggaagagatggaggagaaggagtgggagctcacccaggatgtggtgtccttcgacgtgtataaccacagctggcacagcttgacacagctgcccacaccactgctaggccacagtgtatgtaccactggcaacttcctatttgtccttggaggggagagccttcctggcagcccatctagttccctggctgccggctcaaggtcaatcacagccttagtgcaccgttatgacccgcgcttccacacttggacacaggtgcctgccatgcgagaagcaagggcctacttctggtgtggtgtggtaggtgagagtctcctggctgtcggaggcctgggcatctgtggtgaggcactggcctcagtggagatgtatgaccttcgcagggaccgctggatggcagctggacagctacctcgggcagtacatggtcacgcaggtgctgtcggggatcatggtgttgtgtacatctccgggggcaaggctgggagaggtgaaggaggcacaagtagcctccgggatatgtactcccttagccccggagagagagtgtggagcaagagggcacccatgggcacagctcgctatgggcactaccttgtggtgctgcGAGGTGCAGTGTTTGCCTTTTTGGGGAGATATGAGCCCTTCTCTGAGATTGAACGCTATGACCCCTGCACAGACCAGTGGACTCGGTTGCGGTCGCTACCCTATGACCGCTTCTGCTATGGA

This mutation is a 1657 bp deletion beginning at Chromosome X position 157,818,351 bp and ending after 157,820,007 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50297 AAG CTG AGC AGG CAA AAA TC Common A
50298 GCT GGA GGC ATC CCA TAA C Wild type Reverse A
50299 AGG GGT CAT AGC GTT CAA TC Mutant Reverse A
50300 Fluorophore-1 CAG CGG AGG TGG GGG T Quencher-1 WT Probe
50301 Fluorophore-2 AGC TGT CCA GCG AGG TGC A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50297 0.40 uM
50298 0.40 uM
50299 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.