Protocol 38123: Probe Assay - Gid8<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

>chr2:180714297+180714398 102bp AGTGGATGCAGGAATCTTGG TGAGGCAGACACCTTGCT

Mut = 92 bp

Wt = 102 bp

Fam = Mut

Hex = Wt

WT reverse primer anneals over the nucleotide sequence containing mouse genomic variation rs262999462.

 

Sequence

Wt Sequence (deletions in lower case):

TCTTAGAAAGTGGATGCAGGAATCTTGGCCTGTGGTTTTACCAGACTCCTGTGgatgaggtggcgccagagcaagcacccctgtgaagatgcagcaaggtgtctgcctcacctgcaattgcctctgttgctctggcatcctgacctgtaatccctgccttgatgatgtcacagcacacaatgggctagcagtctgtgaccttcaggttgataggctaagccttttgtatagctgcaaagaaatgacagccttgactattaatcacaaaaatagtaaaaaccaagttcctaagaaactgtggtttatatgtaggatcatttaatgatggttgggctctttagaagtacaaagccttaattcctagttatctttcctatcagtgaagatagttttcttttcagagggctttaaggaagcagcagagaaatttcggatggagtctgggatcgaaccaagtgtggacctagaaacacttgatgagcgaatcaagattcgggagatgattctgaagggtcagatccaggaggccattgccttgatcaacagcctccacccagagctcctagacacaaaccggtatctttacttccaccttcaggtgagtttcagcagtctgatatggctgctagacagacaaattaagcttgtttgcagtggctcagaaagaaatcggccattgtgtgtaaggagacaggctttgagctagctgctgctttatgtactattgcaactagttactttttaaaggggggtgtgtactggtaataagaacacatgttccaccatgttcatagcagccttatttataatagccagaagctggaaagaacccagatgtccctcaacagaagaatggattcagaaaatgtggtacatttacacaatggagtactattcagctattaaaaacaatgaatttatgaaatttttggacaaatggatgtatctggaggatatcatccttagtgaggtaacccaatcacaaaagaagtcattagatatgcattcactgggggctggtgagatggctcagtgggtaagagcacccgactgctcttccgaaggtccagagttcaaatcccagcaaccacatggtggctcataaccatccgtaacgagatctgatgccctcttctggtatatctgaagacagctacagtgtacttacatataataataaataaatctttaaaaaaaaaaaaaaaaaaaagatatgcattcactgataagtggatatttgcccagaaacatagaacccccaagatacaatttgcaaaacacaagaaaatcaagaagagggaagaccaatgggccgatacttcattcctccttagaacagggaacaaaatacccatgaaatgagttacagagacaaagtttggagctaagaagaaaggatggactatccagagactgccccacccagggatccatcccataatcagccaccaaacccagacactattgcatatgccagcaagattttgctgaagggaccctgttatagctgtatgaggctatgccagtgcttggcaaatacagaagtggatgcccacagtcatctataagatggaacacggggcccccaatggagaagctagagaaagcacccaaagagctaaaaggggtctgcaaccctataggtggaacaatatgaactaaccagtacccccagagcttgtatctctagctgcatatgtagcagaagatggcctagtcggccatcactgggaagagaggccccttggtattgcaaactttatatgccccagtacaggggaatgccagggccaagaagcaggagtgggtgggtaggggggcagggtgggggggagggtatagggaactttcaggatagcatttgaaatgtatataaagaaaatatctaacaatcaatcaataaataaatggataataaaaactatacttttatcaaggaaaaaattaaaggggtgtatgtgtctgtgtgtgtgtgtgtgagagagagagagagagagagagagagtatgaatgaatagtgtacatatggaaataagaggcattccttgggagtccattctttctgcccttacatgggctctgggcatacttgcacaacgagcactttcactgtggcagtctcaccaatctctccttttgcttttaaaatggaaatgtgccaggcgtgatggcgcacacctttaatcccagcactcgggaggcagaggcaggtggatttctgagttcgaggtcagccagggctacacagagaaaccctgtctcaggggaaaaaaaaatggaaatgcatcttggagatgctaagaaagtttcttttaagatgcctttcttattttcttttagggtgatggggtggagcccaggcattgcctagatgctaaacaagtactttaacactattagttagacactcaatccacacataagatatacttttgattggagccaagtgttgatggttattcatctaagtgtatgccagccaaacttggttgtctttgctgttgaccatactgggttccacctactgggtgtacaggctggctaatcttgtttgtctttgttttgactagcaacaacacttgattgagcttatccgtcagcgtgagacagaagcagcattggagtttgcccagacacaactggcagagcagggtgaggaaagccgagaatgcctcacagagatggagcgcacactggccttgctggccttcgatagccctgaggagtcgccttttggagacctccttcacatgatgcagaggcaaaaggtaaagggccagtaaaggaagaacagagtgcttagacactcagaggacttgtgaaaagatggtactgtgctgcgcttgtgttggggtgaagcacatggctctgggccaggatactccagtagaacactggcagctccactgatctgtccccttttaccttgtgagagGAAGAGCAGCGCCATTGTGACTGTTAGTGTCTAAAACCCT

This mutation is a 2882 bp deletion beginning at Chromosome 2 position 180,714,342 bp and ending after 180,717,223 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
53056 AGT GGA TGC AGG AAT CTT GG Common A
53057 TGA GGC AGA CAC CTT GCT Wild type Reverse A
53058 GTG GCA AAG GGT TTT AGA CA Mutant Reverse A
53059 Fluorophore-1 CCA GAG CAA GCA CCC CTG Quencher-1 WT Probe
53060 Fluorophore-2 CCT GTG GAA GAG CAG CGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
53056 0.40 uM
53057 0.40 uM
53058 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.