Protocol 45495: Probe Assay - Lrrc28<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=  113 bp

Wild Type = 121 bp

  
>chr7:67619127-67619247 121bp GCATTAGCTGTTGTTATGTCTGTG GGGAAAGTTGCTGTCATTATTCAG

Sequence

Wt Sequence (deletions in lower case):

GCATTAGCTGTTGTTATGTCTGTGTGTTTTGGAAGTTTTGCttatatttgataagttttcaaacagagcttgtattagctggttatctgacaaaatactgaataatgacagcaactttcccctccttttgtcacaggtaccttcactcaaataacatagttgtggttccagaaggtaagcatacttataattcttagtttgaaaacagtgtcatttatattttgctttataaaaatttgatgagccccggaccctgtctttctatctctagctccttaaacctattttggttggtaagttagggaattttaatttaaaggactttcaatttatttgctttaaaatgtaagtccagctaagacagattaaatggagacatctaataagacaataaatggccaggaaaattactagacaaactgctgttgggaaatagtaaactaagtacttggtgcaattaactaagaatttagttacatataaaataatagattctttaaaattcttatgggggggtgttgtggggagggagtgtgtgggaagagggtagcagaggaccactttgtaacttgttcctcagatgtcttttttttgagggaaatctcccctggtctggtactcaccaagcagggtcaacctagaccctctctgcgcccccagctctggttgcaggcatgtgctgctgcgtccccagctctggttacaggcatgtgctgcagagctttttaaatgtagggttttagggtctgggctcaggtttccttgctggcaagacaagcacttaagcagctgagccacttcccagaccctgctcttagtgatgccatctgaggtgggctcctgtgtctccctgctggcttcaaactactctcctgcttcagcctcccaggaccaccccagctgggttcatcttcaaattctttatttagtcgcacattcccccattgtgttaatgtttttacttgttcttaagttaattttatctcaagagatgctttccactttaataacttcattttcaagtcctgtgagtgaaagacagagtcaagagcgaagcgttcgcctaggaacactgagtcacactcattctgcttctcccttccttttctccctgaagccatcgggtccctcgtgaaactccagtgtctggatctcagtgacaatgccttagagatcgtttgcccagaaatcggtggtctgagagcgttgcgtcatcttcgattagctaataaccagctacagttcctacctccaggtaaccctgactgtatagtttctgtgtgtccggaatctgttttcagtaccgtgcacaccagcagctggtgttagatgtatttacaagtatctgtttaaggatggaacgaaggactgtaggtgaaacctgaccaagcgttctgagaacccggtgtaaatgtgagaggatagtgtgtgtgtgttttggggccactgctggggttttgatgagtgtccgctcatcactcttctcactgcTGGGCAAGGTGATTGTGTGAAGAGCTGTTGGCTCTGAATTTCACATCAAAGAATGTGAGTAAATTTGGACGC

This mutation is a 1450 bp deletion beginning at Chromosome 7 position 67,617,757 bp and ending after 67,619,206 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65737 GCA TTA GCT GTT GTT ATG TCT GTG Common A
65738 GGG AAA GTT GCT GTC ATT ATT CAG Wild type Reverse A
65739 GCG TCC AAA TTT ACT CAC ATT C Mutant Reverse A
65740 Fluorophore-1 TTC AAA CAG AGC TTG TAT TAG CTG G Quencher-1 WT Probe
65741 Fluorophore-2 AAG TTT TGC TGG GCA AGG TGA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65737 0.40 uM
65738 0.40 uM
65739 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.