Protocol 36768: Probe Assay - Lyar<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

>chr5:38225815+38225920 106bp GGCCAGGTGTGGAAAGTCT TGCACTTCACATGGCTTTTG

Mutant=  106 bp

Wild Type = 106 bp

Sequence

Wt Sequence (deletions in lower case):

 CCAGGTGTGGAAAGTCTTTGAGAGCCACTAGcatgctgtcagcctagtgaccagccctgggtccctctgcaggggtgatgactacaaaagccatgtgaagtgcatcagtgaaggtcagaagtacggaggcaaaggctatgaagccaagacacacaaaggtgatgcaaaacagcaggcatggattcaggtaagggtccctcccatcctactgctcatccctacctgcttcctcctgggggctcagcagatgggagtccctgagacagcacgtcttagagtgcagcagaagtgtcagaacatcaggaaagataaaattgtgctgcatttatatctggttcgtgtttgctctcaagatatctgagggttcctgctaacttccttttggctgtaaacaaaatcaaagtgtacaggtacacttggcagtggtagaggtcctatagcttagccgtgtcagcctgtcacagctgaacaaagtaaacatttttacaaatttaaggaattttccagtgaaaatatgtacaaaagcagcaagccctggtttgcagcaagagaagatgggtgggcagatgtcacctttgtacagactgcattctcctggaaatcagattagttgttcccccactgaatggctcagagtgagtgtgaacttggggtggtggctctctctgaaggtgtatactgctggcctgaccctcatgagggacttgaaggaggagctgcctggttgagtaccacttgggaagctgctgaaacctttaggatgtgtgtagtgctggcgagatggttgagctcggaccctttcccactgagctgtcctgctgacctttacttaatactgtagtctgccatgtgtaagaacccattatcaggtcagaaggcgcctcctctgccaagggtttttgtgttgttttggtttggttttgtttgtttgtttattggtttttcgagacagggtttctctgtgtagccctggctgtcctagaactcactttgtagaccaggctggccttgaactcagaaatccacctgcctctgcctcctgaatactgggattaaaggcgtgtgccaccacgcccggcttgcaaaggtttgtatagtttcactttttaaaatgtagatccttgattcattttgaggtgttttttgtataacataaggtaagcatctaatttccctgtggggatccactatttctgtcatcatttgttgaaaaggggatctgttctcccatagactcttcttgacatccttgttgaaaagccccttgaccatatcctcaggggtttattcctgcattttctgttcttttccagtggtctttgtggcagtaccacactctttagtttattgtagctttgtagtaagttttgaaatttaaaaaaaaaaacatttttcaaaattgttttgttgtttggagtccatcagggttccatgaattagagcacacatatgctagtctttaccagggctttcaggttaacatctaaatgttgtcagtgcatgaatgccgtttttattcttgtaactaactgtccagtgcccagttccttcaccgagttggttatggttctttatgttgtgctgcagatggaattctcctaatgttcacgtctgaattggtttttttgttgtttggttggttggttggtttttgtttttgttttttggtgtataaaaaagcagcttgttctttaagaggtgagtgtgtgtcctgcagctctgctgaatgcgttgttaattctaactggctgtgtcctgtagcactttctacatgcaagaccacattccctcaaatactgcctgtacacgtgtgacctgattttgttctttctgttaggggggctttggcttctttttcttgcttaaccactttggccaggactccagtgctgtgctgaataggagaatgtttgtcactatgcatagatgtaaatatagagtcttagtttaaaatgtgagagccctttggccatgccaaggacttgagtttggcttcatgtaattatgtcacgtgtttattttcttctatcttaaatagaaaattaatgagttaataaagaaacccaacgtcagccccaaggtgcgagaacttttgcagcaaatcagtgcttttgataatgttcccagaaaaaaggcgaagtttcaggtatggatttaaatgactttcctaaaccatggtgtgacccatctgtggcctgcagaggaacagtggctctttgtcttgtgtccacagaactggatgaaaaacagcctgaaagtgcacagcgactccgttctagagcaggtgtgggatatcttctccgaagcatccagcagtgtgagtcaactccactttgccactgcaatatggcagagaactgggaataaggaaatggggtgccatgtgtggggatcaagatggcatttccagggaAGCTCTGTGCTCTCTCCCTGATGGGCAGGAATGATCTGGGAGCGCCTGTGGTGCAGAGACAGC

A 2382 bp deletion beginning at Chromosome 5 position 38,225,848 bp and ending after 38,228,229 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47942 TGC ACT TCA CAT GGC TTT TG Wild type Reverse A
49612 GGC CAG GTG TGG AAA GTC T Common A
49613 AGG GTG AAT GGC TGT CTC TG Mutant Reverse A
49614 Fluorophore-1 TGC TGT CAG CCT AGT GAC CA Quencher-1 WT Probe
49615 Fluorophore-2 AGA GCC ACT AGA GCT CTG TGC TC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47942 0.40 uM
49612 0.40 uM
49613 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.