Protocol 48099: Probe Assay - Tsr1<em1(IMPC)J> Alt 7
Version 6.0

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=  66 bp

Wild Type = 99 bp

 

>chr11:74899215+74899313 99bp GATGGTCCTCCACATCAGGT CACCGTTCCCAAGTCTTCAT
  

Sequence

Wt Sequence (deletions in lower case insertion in between ^^):

TGATTATTTTAAACAAAGGGTCTCACTGTGTAGCCCAGGCTGGCCTAGAACTCAGATCCTCCTACCTCTGCCTTCTGAGTGTTAAGTTAAAAGGCTTGGGCCACCACACCTGGCTCCTCTGATGTTTTCTTTTTTCTTTTCTTTTTTTTTTTTTTTGGGGGGGGGTGGTGGTGTTTCCGAGACAGGGTTTCTCTGTGTAGCCCTGGCTGTCCCGGAACTCACTCTGTAGACCAGGCTGGCCTCAAACTCAGAAATGAGTGCTGGGATTAAAGGCGTGCGCCAGCTCCGCCCGGCCTGCTCTGATCTTTTCTAATGTTTTACGTTACATAAAATTACCTTGGTACTTTTACAAAGCAACAGAAAGGGGAGTAGTTctgccctggggttacagcacctgtatatttgaagatagaatagtgaagcaaatgattgaggtattcttggatctttgtgttgctaggttctggccgagaagaggcagctgggcagcaaggatggtcctccacatcaggtgctggtggtgcctctgcacagcaggatttcccttccagaggcatttaaactgcttcagaatgaagacttgggaacggtgtatttgagtgaacggggaagcacccagagttttatgcttctgtgccctagcttgaaacatcgatggtttttcacatatgcaagaccaggttagagtgtccgatacttttctctcattgtgtttaagtagttgtctctcttgaatatagtgtgctaactatcttttttttttttttatggtgtcttttgtatacaggggatctgcatactctgttagacatggctaaagtcgctgataccattctgttcctcctggacccactagaaggctgggacagtacgggggattattgcctgtcttgcctttttgctcagggccttcccacctatagtaagtgtgctgtattggataccagcaaagaaaacagctctggtctagtcatgcaggcacagtgtttaagattaagtttaggagtaaatcaatgggttgtattCTGCTGGTGTGGGCTA^aggtata^TACTTGGTAAACATTTTCATATATTTTACTCTTTTTATTTATTTATTTATTTGAGACAACAAGAGTTTCTTTTGGGCGGCCCTGACTATCCTAGAACT

 

This mutation is a 651 bp deletion beginning at Chromosome 11 position 74,899,096 bp and ending after 74,899,746 bp (GRCm38/mm10). There is a 7 bp insertion (TATACTT) 16 bp after the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
67656 GAT GGT CCT CCA CAT CAG GT Wild type Forward A
67657 CAC CGT TCC CAA GTC TTC AT Wild type Reverse A
72600 Fluorophore-1 CCT CTG CAC AGC AGG ATT Quencher-1 WT Probe
72601 CCT TGG TAC TTT TAC AAA GCA Mutant Forward A
72602 AGT ATA TAC TTT AGC CCA CAC C Mutant Reverse A
72603 Fluorophore-2 CAG AAA GGG GAG TAG TTC TGC T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
67656 0.40 uM
67657 0.40 uM
72601 0.40 uM
72602 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.