Protocol 35980: Probe Assay - Zyg11b<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

>chr4:108266719-108266835 117bp TAGGGAAGCACTGGGACGTA CCCAAAAGAGAACTTTCCAAGA

Mutant=  114 bp

Wild Type = 117 bp

Sequence

TTTGGTATCCAGATTGATAGAGACCAGACCTCATAATACCACACTTCTAGAGCTGAGAAGAGAGCATGGAGAATCACCATTGGCTCTCGGGTGCCTAATCCTACAGCGCCTTAGCGCTACCTGGCACGTGGTGCTGTCTGATAGTAGGGAAGCACTGGGACGTATCGCTGCCTAT^G^gtatctaatagtgaatctccgcaatgtctgttccattctgattctgctgttactagctccataatcttggaaagttctcttttgggtcttagtctagacaataatatcttcatctcaccaaattgtatcaagtaagtaaaatgcagctgcatggaattatgttagtgtggtgaaatttttgctcttctttgtctgtgattttgaagttaaaatgctgcttatttttccccaggtctcctgaatgatggaactgtgggaattttcagaggtaaccagatgcgcctaaagcgagcttgcattcgcaaagcaaagatttccgctgttgctttccggaaagccttttgccaccacaagctagtagaacttgatgccacgggtgtgaatgctgacatcactatcacagacatcataagtggccttggcagtaacaagtggatccagcaaaatcttcagtgcttagtactaaactcattaactctgtccctcgaggatccatatgagcggtgcttcagccggctctctggtcttcgagctctgagcatcactaatgtgctcttttacaatgaagatctggctgaagttgcttcgctgcctagactggagagcctggatatttctaacacctcaatcacagatatcactgccctgctggcctgcaaagacagactcaagtctctcaccatgcaccatctgaaatgtttaaaaatgactactacccagatcctggatgtcgttcgggagctaaaacatctgaaccatcttgacatctcagatgataaacagttcacgtcagacatagcccttcgattgttagagcaaaaggacatcttgcccaatcttgtgtccctggatgtttcagggagaaagcatgtgacagacaaagctgtcgaagcctttatacagcagcggcctagcatgcagttcgtagggttgctggctacagatgctggatactctgaattccttatgggcaaaggacacttgaaggttagatattaaaaattgtattttgcttgactggtcaggtttttttttcctagttccttaagacttcatTAGGGTAAATGTGTAATATTTGAAACACTCTGATACAAGGTATGGAAATAATTGTATAGTGGAGAAAGCCTTACATTAGGGTAATAATCCCAATTAAAAGTCCAGTGGGACCAAGCATGGCGAATGCTTTTAATCCAAGCACTCACAGAGCAGACGAAGGTAACTTCCTGCATGTACATAGCCTGGTGTACATGG

 

This is a 1055 bp deletion beginning at Chromosome 4 position 108,265,749 bp and ending after 108,266,803 bp (GRCm38/mm10). There is a single bp insertion (G) at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47463 TAG GGA AGC ACT GGG ACG TA Common A
47464 CCC AAA AGA GAA CTT TCC AAG A Wild type Reverse A
47465 CCC TAA TGT AAG GCT TTC TCC A Mutant Reverse A
47466 Fluorophore-1 AGT GAA TCT CCG CAA TGT CTG Quencher-1 WT Probe
47467 Fluorophore-2 CTG CCT ATG GTA GGG TAA ATG TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47463 0.40 uM
47464 0.40 uM
47465 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.