Protocol 38190: Probe Assay - Lrrc4b<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

>chr7:44460712+44460827 116bp CAGCCTTGGGTGCTTAATGA CATGCACCAATCGCTCACT

Mut = 100 bp

Wt = 116 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GCCAGCCTTGGGTGCTTAATGAAACCCTGTCTCCGAATACAAAgtaaaaagacagctgggaatgaaggataatggtagagcacctacctagaatcccccagtgagcgattggtgcatggctcagtggctattgaccatctagaatcctcagtcacagagtccctgtccagcatgtgagaggccctaggtcttgtccttaggcccacatactgatcgcagagtcgcgttcacctgctgtgaactggagcagcctgctgctcccacgcttgtcaggtgcactgacactggtctccccgcaggtgatccgcacggacacattcaagcacctccggcacctggagatcctgcagctgagcaagaacctggtgcgaaagatcgaggtgggagcgttcaatgggctgcccagtctcaacacactggaactcttcgacaaccggctgacaaccgtgcccacgcaggcctttgagtacctgtccaagctccgggagctgtggctgcgcaacaaccccatcgaaagcatcccatcctatgccttcaaccgtgtgccctcactgcgccgcctagacctgggcgagctgaagaggctggagtacatatctgaggcggccttcgaggggctggtgaacctgcgctacctcaacctgggcatgtgcaacctaaaggacatccccaacctcacagcacttgtgcgcctggaggagctggagctgtccgggaaccggctggacctaattcgccctggctccttccagggcctcaccagcctgcgcaagctgtggctgatgcatgcccaagtggccaccatcgagcgaaacgccttcgacgacctcaagtcgctggaggagctgaacctgtcccacaacaacctgatgtcgctgccgcacgaccttttcacgccccttcaccgcctggagcgtgtccacctgaaccacaacccctggcattgtaactgtgatgtgctgtggctcagctggtggctgaaggagacagtgcccagcaataccacatgctgcgcacgctgccacgcgcccgccggcctcaagggccgctacattggcgagctagaccagtcgcacttcacctgctatgccccggtcatcgtggagccacccacagacctcaatgtcaccgagggcatggcggctgagctcaagtgccgcacaggcacatccatgacgtcggtcaactggctgacacctaacggcacgctcatgacgcacggttcctatcgtgtgcgcatttcagtgctgcacgatggcacactcaacttcaccaatgtcaccgtgcaggacacgggccagtacacgtgcatggtgacaaactcggctggcaacactactgcctcggccacgcttaacgtgtcggccgtggaccccgtggctgcgggagggcccggtggcgggggcccagggggtgggggcggtgcagggggcgcgggcggatacacctacttcaccacggtgaccgtggagacgctggagacgcagccgggggaggaggcccagcagccccgaggtactgagaaggagcccccggggcccacgactgatggcgcgtggggcggcggccggcctgatgctgcagccccggcctcggcgtccaccacggcaccggccccgcgctcctcgcggcccacggagaaggctttcacggtgcccatcacggacgtgacggagaatgcgcttaaggacctagacgatgtcatgaagaccaccaaaatcatcatcggctgcttcgtggccatcaccttcatggccgccgtgatgctggtggccttctacaagctgcgcaagcagcaccagctgcacaagcaccacgggcccacgcgcactgtggagatcatcaacgtggaggacgagctgcccgccgcctccgctgtgtcggtggctgcggccgccgcggtggctggaggcgcgggcgtgggtggggacagccacctggcccttccggcgctggagcgagaccacctcaatcaccaccactacgttgcggccgccttcaaggcgcactacggcggcaacccgggaggcgggtgcggggccaaaggccccggcctcaactccatccacgaacctctgctcttcaagagcggctccaaggagaacgtgcaagagacacaaatctaaacccaggcgcacgggacgcgggacgcctgcccgagatgctcctgcttgccgagcaaccctggcggcgcctgcgggtcctgcaatgggaaaggacctggggaccaggagaggtggccccgccctccgccttgccgcgtcctctgccctcctgccctgcccgccaccttccaggggttgaggagcttcttcgaggggtacccttttctcctcaccccaacaaccctccttttatggctctttttttttcttttcttttcttttttttttttcttttgcagtttgacgcctgtccttccctcctactccttcaccctcaaaacacaagagacaggcaacttcaccagagcccagggggcaactccgacaatgcccccaacagcgagctccactcgcggccctggcaggcccaccccaccccacagactcttttgatactgaagggaggtttgcgtcaatcactacctgctctgtaattactttaaaaaaaaaaaaaaacacggaaaaagtaaaaaaaaatttttttgggggggggggcatgaaagtattgaagagaaacagggcacaaagctcctgagggtgggacgaggagaggataggcagccagcgatgctgtcctcccaggaggggagagtggatactccaaagttacagctctgggactctctatccccgagattccagagaggaacaggctcacccagcacatagcagcacggctccatctccccatGTGCTGGGGGTTCAGTAGCTGCCCTTCTGTGTCTCTGTCTCCATCCCCATGTCTAGCTGTTGTTCCTTTCTGACTCTGGTGCCTCCCCCATCACACACACAGCA

This mutation is a 2813 bp deletion beginning at Chromosome 7 position 44,460,753 bp and ending after 44,463,565 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
53247 CAG CCT TGG GTG CTT AAT GA Common A
53248 CAT GCA CCA ATC GCT CAC T Wild type Reverse A
53249 CAG CTA GAC ATG GGG ATG GA Mutant Reverse A
53250 Fluorophore-1 CAG CTG GGA ATG AAG GAT AAT G Quencher-1 WT Probe
53251 Fluorophore-2 CAA AGT GCT GGG GGT TCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
53247 0.40 uM
53248 0.40 uM
53249 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.