Protocol 36170: Probe Assay - Hps5<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

>chr7:46788654-46788765 112bp GTGGAAAATGCTGATTTGAGG GAACCGTGAGACACATCC

Mut= 107 bp

Wt= 112 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TAAACACTTGTAAGTGAGCAAATGATTGAAGTAGCTGTTTTGTCCCTGGTTTCCTCCACTGTAACTAGCACTGGAATAACCTGGTCAAGTTATGAAAATATGAACTGTTTAATAACTGGTTTTAACAAAGAAAAATTACACATTTATACAATTGAGCCTATTCAGGTTTTGGAGATTCAGCTAAAAGTTTAAAAGGGAAATGTTCTTTGTCTGGCATATTTCAGTATTCTAGGATTATAAAGGAGTGCTGTTACATTGTGTCccgggcaggggagagaaggaaggaagtgagcacagacctgcaatacttagttcaagtggctaggttatttttaagtgttgggaatgcagcctaggttctcgtgcatgccaggtaggtgtcctgtaaccgaggtgcatccccagtgcatagcagatctcacgattgtctcctcatgctcttagtgtaccagcatagctgtgtctcggaaatggttggcgttaggcagcacaggaggaggactcaatctcattcagaaagatggctggaagcaaaggctgtttctctctcaccgggtaagaccaaggacattcacccactcccccccaatgtaatcaattatttagagtaaaagaagaatgaaaacgaactattcttgtctgatgttctaggaaggggcaatctctcagattgcctgctgctctcatgatgatgattatgttgctgtagctaccaggtaagaaacccttaaagctgacatgtgtgcttgtcaggatgaccaaccatctcatctcacccagcactgtcctggtgtcagttctgaaagtcacatcctggggatccaagtctgctcagtgcccaagtcacctgggaggggtcgttggtcaccctggtgtttgtggaaaatgctgatttgaggcaacaacatctaaaaggtcttaagagagaaatggaagagtcgtcacctctcccTGAGGAGCTGGTACACGTCTGGATGTGTCTCACGGTTCTCTCAGGGCCTGCTTCTTTTCTCAATCTTCATGCTTAAGCCTGTGCTATCTCTAATCTTGCTGAGAGAGAGTGGGAGCAGAGGATGTGCAAAACATTTGAACTCTTAAGTTCTCCTATTTTATTGGTTTGATGACCAAAACAAGGAAGTGAATACTTTGACTTGTAACTCCTTCCCCTAAGAACAGGAGTTTATATTGTATTTAATATGGCTAATATTT

This mutation is a 690 bp deletion beginning at Chromosome 7 position 46,788,692 bp and ending after 46,789,381 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48001 GTG GAA AAT GCT GAT TTG AGG Wild type Forward A Wt F
48002 GAA CCG TGA GAC ACA TCC Common A Common
48003 AGG GAA ATG TTC TTT GTC TGG Mutant Forward A Mut F
48004 Fluorophore-1 AGA GAA ATG GAA GAG TCG TCA CC Quencher-1 WT Probe
48005 Fluorophore-2 AGT GCT GTT ACA TTG TGT CTG AGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48001 0.40 uM
48002 0.40 uM
48003 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.