Protocol 35710: Probe Assay - Exoc3l4<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

>chr12:111425084+111425173 90bp GAGAGGAGGTAGCTGGTGGA GGAAGTCAGGACTGAAAAAGCA

Mut= 90 bp

Wt= 90 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GATTTCATTACGGGCGGTTGTGAGCCACCATGTGGTTGCTGGGATTTGAACTCAGGACCTCTGGAAGACCAGCCAGTGCTCTTAACTGCTGAGCCATCTCTCCAGCCCCATGACTTTAGACTTTAAGCCAGGTTAGAGAGAGGAGGTAGCTGGTGGAGCATCAGAGGTCCGCCTGGgtcctaacaactccctcatcccggaatcttgctttttcagtcctgacttcctgggcgccccagacttggcgctgcccaccgagccgctgcccccacttctagagcctgctctgtgggctcgactggaaagtgactacactagcttcctagaggtgaggctggggaggccccgggctccagaggttgggtcctacaggaggtttgatgtggaggataggggttgtgctggcagtggtcctgtaagcgatggaccatgactggagtccggtgacgctagtccttgaaggcgcaaaatggagtttggtaggcagggggcgcccttaccttcctcctggaaagcttctcaccacaggccactcctaaccctacagaccaagatcacaagctgttttgacagcatcttgcagctggaacagaatcgctgggaagccgacgaggaccgggaagtactgcagggcctctaccacgcgcccctgtccatcgatgtgcacatggtgcgcccgggagggggtgaaggaagggctatgttcaggactcaggctcatgtcggagatccctcagagctaagttttactgagagaggaacctgtggAGTGATCTAGACTCTTGGGGCACATGGCCAGGTTAAATCACAGCTCTCGGGTCTGCCGGTGAATTCTAGTAGCTGTGTGGAAGGGGGGGAGGTGACTCTCACATTGGGGCCACTGAAAACCTGCAGCGATGGGGTGGGCTGGGTTCGGGACTCAACTGCTCTCAACATGTGGCATCCCTGGCAGCTGGTAGCTGAG

This mutation is a 592 bp deletion beginning at Chromosome 12 position 111,425,123 bp and ending after 111,425,714 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46828 GAG AGG AGG TAG CTG GTG GA Common A
46829 GGA AGT CAG GAC TGA AAA AGC A Wild type Reverse A
46830 CCC GAG AGC TGT GAT TTA ACC Mutant Reverse A
46831 Fluorophore-1 AAC TCC CTC ATC CCG GAA Quencher-1 WT Probe
46832 Fluorophore-2 CCG CCT GGA GTG ATC TAG ACT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46828 0.40 uM
46829 0.40 uM
46830 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.