Protocol 37135: Probe Assay - Tsen54<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

>chr11:115820986+115821119 134bp CCTGATAAAGCCGATTGTCC AGCCTTCTCCCACACCTCA

Mut = 120 bp

Wt = 134 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GAGTGAAGGTGGGTGACTAGGCCCTTGTGGCTGTTCCCAGGTACCCTTCCTTCTGCTATGGCTCTGCCTCTGACCTTTGCCTCATCCTCTTGGCCAAGCTCTCCCAAATCCCATCTACGgtggcggcagcctgagtgaacggacccgcctcctgttctcacccacagctctgtcgtgtcaccttacgagcggcagcttaatttggatggttatgcccagtgcctggaggatgggtctggcaagaggaagaggagttccagctgtcggtaatccacctgctgctgccacccctgggagcacatgggcttaagaaggggcctgaggacacagccgagggtatacctcagttccagtggaatgtacgtgtcttacggacagtaacaagggacaaatgtgctcttttttctcccgtgtctctaaggtctgttaataagaagcccaaggtcctgcagaactccctgccgcccgtcagcctggcagcctccagctcacctgcctgtgaccagagtagccaatacccagaggagaagtctcaggactcaagccccaggcagggctcagagctccccttgcagttcctggggtcctcagagccttgctctgatctggccagggaagacgtggggtgtgaccgagagagtcacaaaatagagaatggagctaagggaacccctaagctacgctggaactttgagcagatctcattccccaacatggcctctgatagtcgccacactttcctgcctgctccagccccagagctgctcccagccaatgtcattgggcgagggacagatgctgagtcctggtgccaaaagctgaaccagcggagggagaagctctcccgtagagaccgggaacagcaagcagtggtccagcagttccgggaggatgttaacgcagatcccgaggtgcggggctgctccagctggcaggagtacaaggagctgctgcagaggcgacagacgcagaagagccagccccggcctccacacctatggggccagtccgttacccccttgctcgaccctgataaagccgattgtccaggtatccctttaccctgagctatacccctataggctgttgggagcagagagagacttctgaaagtggatgcttagacgAGGGCTGGCTAAGGCCTGAGGTGTGGGAGAAGGCTGTGCAGTGGGGTGGGACCTATATAGGCTGTTCCGTGGGTGGTGGTGGCAGCCACTGGGGACAGTTTCCTGATGAGTCATTGACCAGTGATTCCCGTGGGGGATGAAAG

This mutation is a 1008 bp deletion beginning at Chromosome 11 position 115,820,077 bp and ending after 115,821,084 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50627 AGC CTT CTC CCA CAC CTC A Common A
50628 CCT GAT AAA GCC GAT TGT CC Wild type Forward A
50629 TCC CAG GTA CCC TTC CTT CT Mutant Forward A
50630 Fluorophore-1 CCC TGA GCT ATA CCC CTA TAG GCT G Quencher-1 WT Probe
50631 Fluorophore-2 TCC CAA ATC CCA TCT ACG AGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50627 0.40 uM
50628 0.40 uM
50629 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.