Protocol 37957: Probe Assay - Klhl9<em1(IMPC)J>Alternate 1
Version 2.0

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

>chr4:88721927-88722011 85bp GCTTCTCCCATGAAGGTGTC ACCGCGTAGTTCCAGACTTG

Mut = 104 bp

Wt = 85 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

TGCGGCCTGCAGCTCGCAGCGAGCTCCGGCTTCTCCCATGAAGGTGTCTCTGGGtaacggcgacatgggcgtctctgcccacctgcagccttgcaagtctggaactacgcggttttttaccagcaacacgcacagttcggttgtattgcaaggctttgatcagctcagaatagagggcctactttgtgacgtgacgctggtacctggcgatggagaggaaatcttcccggttcatagagccatgatggcgtctgccagtgattatttcaaggccatgttcactggaggaatgaaagagaaagatttgatgtgcatcaagcttcatggggtgaacaaagttggtctgaagaaaatcattgattttatttatactgcaaaactgtctcttaatatggacaatcttcaggacactcttgaagctgccagctttctacagatcttgccggttttggatttttgtaaagtatttcttatatcaggcgtctctttggataactgtgtggaggttggccggattgctaacacctacaatcttatagaagtggataaatacgtaaataatttcatcctgaagaactttcctgccttgttgaatactggggagtttctgaaactcccctttgaacggcttgcctttgtgctttccagcaatagtctcaagcactgttcggaacttgagctcttcaaggcagcctgccgctggctgaggctggaagaccctcggatggactatgccgcaaaattaatgaagaatattcgctttccactgatgacaccacaggatctcatcaactatgtgcagacagtagatttcatgagaacagacaatacctgtgtgaatttacttttggaagcaagcaattaccaaatgatgccgtatatgcagccggtgatgcagtcggacagaactgccattcgatctgattctactcatttggttaccttaggaggagttttgagacaacagctagttgtcagtaaagagttacgaatgtacgacgaaagggcgcaggagtggaagtcgttagcgcccatggatgctcctcgataccagcatggcattgctgttattggaaatttcctttatgtagttggtggccagagtaattatgatacaaaaggaaaaacggctgttgatacagttttcagatttgatcctcgttataataagtggatgcaggtggcatcattaaacgaaaagcgcacattctttcacctgagtgccctcaaaggacatttatatgcagttggtgggcgcagtgcagctggtgaactggccacagtagaatgttacaacccaagaatgaatgaatggagctacgttgcaaagatgagtgaaccccactatggccatgctggaacagtatatgggggtttaatgtatatttccggaggaatcactcatgacactttccaaaatgagctcatgtgttttgacccagatacagacaaatggacacagaaggctcctatgactacagtccgaggtctgcattgcatgtgtacagttggagacaagctgtatgtcatcggtggcaaccacttcagaggaacaagtgattatgatgatgtcctaagctgcgaatactattcgccaactcttgaccagtggacaccaattgctgctatgctgagaggtcagagtgatgttggagttgcagtctttgaaaacaaaatctatgttgttggtggatattcttggaataaccggtgcatggtagaaattgtccagaaatatgacccagaaaaagacgagtggcataaagtttttgatcttcctgagtcgctgggtggcattcgggcttgcactctcacagtttttccacctgaagaaaaccctgggtcaccttctagagaatcacctctttcagcaccttcagATCATTCTTAGATCCAAGGTGTAATACCTTTTCAGTGTATCATGGGTGGTCTTGTTCATCCCCTTGTATTGTTTCTCCTTATTGCTACAGTTAATAGACAAAAAGGTAACTTAGACCCT

This mutation is a 1826 bp deletion beginning at Chromosome 4 position 88,720,160 bp and ending after 88,721,985 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51077 GCT TCT CCC ATG AAG GTG TC Common A
51078 ACC GCG TAG TTC CAG ACT TG Wild type Reverse A
51080 Fluorophore-1 ATG GGC GTC TCT GCC C Quencher-1 WT Probe
52620 GGA GAA ACA ATA CAA GGG GAT G Mutant Reverse A
52621 Fluorophore-2 CCT TTT CAG TGT ATC ATG GGT GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51077 0.40 uM
51078 0.40 uM
52620 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.