Protocol 38192: Probe Assay - Lcorl<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

>chr5:45775455-45775623 169bp TGAAAGGCAGAGAAAATGAACA ACTGGCCCTGAGATGATAGC

Mut = 162 bp

Wt = 169 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

TTTTATGAAAGGCAGAGAAAATGAACATTAAAACAATTTTTATATATTAATTGGATctcaggtttaaatcaaaagtaatgaggtatgttactttcttataattttaggattgtataccatctcttgattcttcacagtcaacgccaacagaggagctatcatctcagggccagtcccacactgataagattgaatgccaagcagaaagttacctaaatgcactctttcgaaagaaaggtaatactggcatgctttgtcatttaaagtttgcattgtttaggtttttcttatatatcagtccctactttattgttaacgacttctttttttgtcttactagagccaataagagtgtcagctctctagtgctagccatttgttatccgcctcctgttctcaacttcttcatgggcacctctcaaaatattttttgtgcttcccactttcattattttcttgctattctcaagtctttaaaaagtgctggacgatgaagaagctatagataggttatattgcaggcctatccctagtactgtatctcttacatcatttcccagctggtgtaccacttaagtatttgtcagcttgagtatgcgtgcttgacattaaaaagtctgttctgtatttgtctctCTCTGGCGTCTTTCAACCAAATGGGTCTTGTTTCACTTATTCCTCTACTTGTTACATATCTATAAGGAAAAGTAGTTTTTATATTTAAACTGCTTGGGAAGATTATAG

This mutation is a 580 bp deletion beginning at Chromosome 5 position 45,774,993 bp and ending after 45,775,572 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
53258 TGA AAG GCA GAG AAA ATG AAC A Common A
53259 ACT GGC CCT GAG ATG ATA GC Wild type Reverse A
53260 CGA CTA TAA TCT TCC CAA GCA Mutant Reverse A
53261 Fluorophore-1 CTT GAT TCT TCA CAG TCA ACG CC Quencher-1 WT Probe
53262 Fluorophore-2 ATC TCT GGC GTC TTT CAA CCA A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
53258 0.40 uM
53259 0.40 uM
53260 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.