Protocol 44218: Probe Assay - Hemk1<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

Mutant=  121 bp

Wild Type = 115 bp

  
>chr9:107331589-107331703 115bp ATCATTTGGCTTTGGCTCTC AGAGGGTCGATCAGACTAGGTG

Sequence

Wt Sequence (deletions in lower case):

ATCATTTGGCTTTGGCTCTCCCCCACCTGTCTTCAGTAGGAGCCAACTGCTTGGACTTCCTTGTAGaggctcctcaggagctaagatgatcagcacctagtctgatcgaccctcttccttccaggaactggttgaatgggttttagaggaagtggcccagaggccccatgcagtgagagcccaggatggtcccctcattctggaggtgggctgtggatcaggagccattactctcagcctgctgagccagctccccaaggtaggctgtctctgcatccctctaccatgccttgagccattgagatggtcccttctggtattctgtcccccacatccctctgtaagaagcattgagggaaagtggccatataaggaagatgtgcccagcccagcatgatcacagcctctaacttccatccttctccatctggctagagtctgaccacaggaaaactttgttatgtattcctttccaagagggagctaggggaagattgtagacagacatctcccagaaattcacctctcttcctttgttcttggggcagagtcgagttgttgctgtggataaggaggaagctgctgttagtctgacccatgagaatgctcagaggtacacgggctggaccttccagaaggccagcactggggaggtggactctgggacctgggcctgacctttattctagtacaaacccactagtgtttgacttgattctcgagggagagacacaatgaggaagcctggcaatggtcccatgggtttgcgagtggtggactgtgagccgtggttatgctgagccaagaagggctccctttccctcctggtccttgtagccaaggtttgcaccctatgctcagcactatctccccatctccaggcttcagctgcaggacaggatccggatcatccaccttgatataacttcaggtagatcccctgtacatcttggggtgggcgctgctgctggtggtgaggcactgcagaaatggggtaaggtaagggtttcccaataatcaaacctgtggctcatctcacaccagccctgctgagcttgctgcgggctcacaaggtgctggtgtctccttggctgacccgacccatgaagggactagccatgaggtccggcctttttctcctcttctctcctctcctctcctcccctctcctctcctctcctcccctcccctcccctcccttcccctctcctcccctcccttcccctctcctctcctctcttttcttgtctgtctttctttttatttcattttttgtttgtttgtttgtttgtttgtctttcaagacagggtttctctgtgtagccctggctgtcctggaactcactctgtagaccaggctggccttgaactcagaaatctgcctgcctctgcctcccaagtactgggattaaaagcctgtgcaaccactgctcaaccacaagcaataagggatcttctgggtcccctatgcctgataaatttgactaggacttatttcctgggaagaggtaaacatgttcaaagcaaagcagttgagagccaagtatggtggcagtgaggattcctgtgaactcagggtctgtctggggaacatggtgacttctagaccagcgtaggatataagcagaacatccccaaaaatcaaaataaacagcaacaataaaggaactttaggaccaaaatgcacgtgtggcctcctgcccacatggcctggatgaggtggtccagcagcgcctggtctgtgcaggttttgcagtgtcttcaggtttccagctccaattctgattcttttgttatccatggagcccccaagcgttgtatgagtgttagacacctgccttagcatctggtacaagtgggatccaaaagcagccataaggggttgtctgagggcagcaagtctgccctccctgtacatccagactatattggttgtaaagcagagaatggtaaggccaaggggctgcagagctgaggaaccctgcctttgcctttgcaatcaaggagggcttactggaggtggcacttgaatttggcttaatgaggaggccttgtcagggaggagagagagaacatggatattctaggtgaacagctacatgtctaaacatgtccactccggccagcctgctaactccggctactaatcctcgagtctggcttattaggtctaaagagagatttgaagagagggactacctaagggacaaggtggcaagcggatctgtcacctttcacacactatgcatgtatcttcctctttgctcacagaggggtgctgcacacaccttttgccctggagccccttggacttggtggtcagcaaccctccctacatcttccgcaaggacatggaacagctggctccagagatctgcaggtacagggtgggtgggacagcagagaagctctagcaggattggttggggtggaggcccttctatcaggagaagatggtgcagtatggaacagCAGGGTTGAGGGGTACACTTCAGAGCCTGCAGCCTTCCTGGGGAAGGTGTTACCT

This mutation is a 2406 bp deletion beginning at Chromosome 9 position 107,329,232 bp and ending after 107,331,637 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65029 ATC ATT TGG CTT TGG CTC TC Common A
65030 AGA GGG TCG ATC AGA CTA GGT G Wild type Reverse A
65031 AGG TAA CAC CTT CCC CAG GA Mutant Reverse A
65032 Fluorophore-1 AGG CTC CTC AGG AGC TAA GAT G Quencher-1 WT Probe
65033 Fluorophore-2 CTT CCT TGT AGC AGG GTT GAG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65029 0.40 uM
65030 0.40 uM
65031 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.