Protocol 34883: Probe Assay - Slc27a6<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

>chr18:58582442+58582535 94bp AATAAGAGGAAGGCCACCCAAG CTGACCCTGAAGTGCTCCTG

Mut= 101 bp

Wt= 94 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TGCACATTCAATGCTATAGTAGTACACATGCAATTACTGATTTTGATTTCCATTCGACAAATCACAACAAGTACATTTGCTATCAATACTGCATGACTGCATTAAGACACGGATCTTAAAGGTCATGTTTCCAAATGAGTTTGTAACcagtgaccgaggatgtggcatgaaagcctggatctgttaggacatcactatcagctgtgtgccacgttaacttaatgccttttatcaatctcttcacataggcgccacgtgtgtgctaaagaagaaattctccgcaagccagttttggaatgattgcaagaagtacaacgtgactgtgtttcagtacattggggaactttgccgttatctgtgcaaacagcctcaggtaagagagtgttgctggagatgctaagggcttcagacagaatatgctttctttgcttttgctgtacaatgctccagaagagaatctatcaaacacacacaggtgttcatagtatacaggatagaagaaatataaaagtccaagaaaataagaggaaggccacccaaggaagtctcaataatacgctttgtcctgcagCAGGGCCAGGCAGATCCATAGTCAGGAGCACTTCAGGGTCAGGCCAATTCCAGTTTGATTGTGAAATTAGATGCATAGATTTACAGAAAAACAATTCCAAGTTGAACGCAGATAATGCCTTCTATATAAACCTG

This mutation is a 424 bp deletion beginning at Chromosome 18 position 58,582,070 bp and ending after 58,582,493 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44695 AAT AAG AGG AAG GCC ACC CAA G Wild type Forward A
44696 CTG ACC CTG AAG TGC TCC TG Common A
44697 CTG CAT GAC TGC ATT AAG ACA C Mutant Forward A
44698 Fluorophore-1 CAA TAA TAC GCT TTG TCC TGC A Quencher-1 WT Probe
44699 Fluorophore-2 AAT GAG TTT GTA ACC AGG GCC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44695 0.40 uM
44696 0.40 uM
44697 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.