Protocol 33181: Probe Assay - Lrrc41<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

>chr4:116088172+116088261 90bp ACGACACAGGTGACACAAGG GGAAGGAACATACCATATTAGTGC

Mut= 87 bp

Wt= 90 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case and insertion with carrots ^g^ (GCACAAATCTAGGTTT insertion):

 CTGCCTTCTGCTTCCCAAGCGGTGCACACTTTTAATCCCAGTGGTGGAGAGGAACAGATCCCTTCAGCTTCTGGCCTGGTAGCCTAGCATATTTGCCAAGTCTCAATCCAGTAAGAACCTCTTACCAAAAAAGGTGGACAGCACCTAAGGAGCTATGCTGGACGACACAGGTGACACAAGGTATCACTTGTCAG^acaagggacactactgtagttaccctacagaaagcactaatatggtatgttccttcctgtttatatcccttgcttatctgtaaaatggggccaccagtaatgcatacctcaaaggtgtgaacactaaataaatttgcagatttaaactaaagcctatctcagtgagtactcagaaatgtttgctacaggaaagaaacttagagggcatttgacaaatttctggtgttttctttgctctttagagtgtgacttgctggcgagccaagtttatggaggcctttttttcccatgttctacgtggaaccattgatgtgtcttctgataaacgtctttgtgatcagcgcttctcacctctcctgcacagctcccgtcatgtccgacagctaaccatctgcaacatgctgcagggagcaactgagctagtggcagagcccaaccgtagggttctagagaccctggccagttctctgcataccctcaagttccgccacctgctgttctctgatgtggctgctcagcagtcacttcgacagctgctgcatcagctcatccaccatggggctgttagccaagtgtcgctgtactcctggcctgtgcccgagtcagcccttttcatccttattcttaccatgagtgctggcttttggcaaccaggcccaggtagcttgccttgccgcctctgtggtgaagcctcccgaggccgggccccatctagagatgaagggtccttattgctaggctctcgcaggcctcggcgtgatgcagcagagcgatgtgctgctgcactaatggccacccggcggaagagtgaagtcaagcagatgcccagggctgtacctcccactcgtgtaacacgccgcagcacacaagagagcctggcaataggtggaacagactctaagttgtacctcccagccacctcttatgaggcttctggcacaaaacagccatctgctccagctgcagcctctgcctcctcttccacatcatccaaacgggctccagctagctcagcctcacagcctaagcccttaaagcgtttcaaacgagctgcagggaagaaagggccccgcacccgtcaggggtctggtgcagagtctgaagacctgtatgactttgtttttattgtggctggtgagaaggaggatggtgaagagatggagattggggaagtagcttgtggagctctggatgggtcagatcccagctgtttggggctcccagcactagaggcatcccagcgattccgcagcatttccaccttggagctattcacagttcctctctccacggaggcagctctgacactgtgccatcttctgagttcctgggtatcactggagagccttacactttcctacaatggtgagcacctgaaggaacagggctgggcttgcatctctcaaaagcactgtgcctaatcaag^AGTGGAGAGTATGATTAAGGAGGTGGAAATGTGGTTATTTGTGTCTCGTGGAGGCAAAAGAGAAATACAAAAGACACACAGTGTTAGGAAAGGAAAATGAGGTCTGTAGTCTGGATTTACTATTGGAGgaaaggccaagGGGCTGAGAAGTCAGATAGATATAGAGGGCAATCCAGACACAGACAGGGCTCTGAATTTGTATCGCATCAGTCAAATTAAAGCAACTGACCAGGGATTTTTTTAAAG

This mutation is a 1426 bp deletion beginning at Chromosome 4 position 116,088,205 bp and ending after 116,089,630 bp (GRCm38/mm10). In addition, there is an 11bp deletion (CTTGGCCTTTC) 128 bp before the exon deletion, and a 16 bp insertion (AAACCTAGATTTGTGC) at the deletion site. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40844 ACG ACA CAG GTG ACA CAA GG Common A
40845 GGA AGG AAC ATA CCA TAT TAG TGC Wild type Reverse A
40846 ATA ACC ACA TTT CCA CCT CCT Mutant Reverse A
40847 Fluorophore-1 AAG GGA CAC TAC TGT AGT TAC CCT ACA Quencher-1 WT Probe
40848 Fluorophore-2 CAC AAA TCT AGG TTT AGT GGA GAG TAT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40844 0.40 uM
40845 0.40 uM
40846 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.