Protocol 35266: Probe Assay - Slc25a48<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

>chr13:56450547+56450694 148bp CCCCAAGACCATGAACTCAG TCCCAACACATCTCCTCACT

Mut= 146 bp

Wt= 148 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TCCCTTTGTCAGCCTGTCACCACAGACTTAGTGAGTTGCACAGTGTTGCCTGTCAGTTTGTCTAAACCCTCCATCCTGTCCTCATCTGCCTTACCCCCAAGACCATGAACTCAGTGAAGGTGAACACTGTTTTGTTATTGATTTGACTCATAGAAAGTAATGAGagagacagacagacagacagacagacagacagacagtgctagctttgcgtgagcagctagtgaggagatgtgttgggacttaacatccccatgactcttgagggctcctcagacattctgcagccacagttctgacactgccatttgggacatgcatatatctgaggccaagactccattgtgcaggaggctcatctttggggctagaatgactgcctgagttgcatgtggccttctcctatcatgccatgtcatctagacccttgctgggggctcttctgtggcatggtggattattaactctgagcctggggagcagggaggtcccatacttggtatgcagagagagaccctgaagagccgtttggcctcacacttcctcccattgggtctccttgccacaggtgtttggcttcttcaagggcatgtccttccccctggccagcatcgccatctataactcggtggtgttcggggtcttcagcaacacccaacggttcctcagcaagtaccgctgtggggaactcgaggctggccctggccgaagtctgtctgatctgctcctggctagcatgctgactggtgtggtctctgttgggctgggcgggcccgtggaactcatcaagatccgactgcagatgcaaacacagccatttcgggaaggtaagaggccgatggagagaaatctggtgtctgaggacccgtgaccctttcccatttgtctggatctgtggtcccattgttcataaccttctctgtgagcaagaccaccctcttccctgctcaccacagggagcatctcattttctcctcttgggagtttcccatgctgcttttgaaggtggctttgacagaggaggaaagagaggagaggggaacccgctgtgtgtcacctttggctgacttgagtgctggaggagacatatttcgattctgtgggttttgttcttggttcagtctcaagtctccatgtctatagaatggaggggctggatctctggtctttcctgtcatagTGTATGTATTATGTATGATGAGTCCCCCCAACCACCCCGCCCTTGCTGGCAGCCTTCAGAATGGTCACTGTGTGAACCCTCTGCACTCTAAGGCTGGGGTTCAGCTGTGGAACATGACAGGAGGCCCACAAGCTCACTCAGGAGCATCCTGGCC

This mutation is a 1016 bp deletion beginning at Chromosome 13 position 56,450,617 bp and ending after 56,451,632 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45651 CCC CAA GAC CAT GAA CTC AG Common A
45652 TCC CAA CAC ATC TCC TCA CT Wild type Reverse A
45653 TTC ACA CAG TGA CCA TTC TGA AG Mutant Reverse A
45654 Fluorophore-1 ACA GAC AGT GCT AGC TTT GCG Quencher-1 WT Probe
45655 Fluorophore-2 AGT GTA TGT ATT ATG TAT GAT GAG TCC CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45651 0.40 uM
45652 0.40 uM
45653 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.