Protocol 31824: Probe Assay - Igsf9b<em1(IMPC)J> Probe
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

Mut= 98 bp

Wt= 98 bp

Fam=Mut

Hex=Wt

>chr9:27317023+27317120 98bp CACTGGGAAGGAGCTAAGTTCA CTGCTGGAAGGGACAATCTG

Sequence

Wt Sequence: (Deleted region is in lower case)

 

ATCCCTGGATATGGCAGTCTCTAGATGGTCCATCCTTTTGTCTCAGCTCTAAACTTTGTCTCTGTAACTCCTTCTATGGGTGATTGAATGAAAGCATTTCTAAAAGTTCTTTCAAGCTGATTATCCAAACAGGATAGAATGTTAATCACACCTTCAGTGCTTGGGGGAGGATAACCGCTCATGTAATCAGGGGAGAATTAAGAAGGTAGAAGCCTGTAGAATTCACTACTCACCCCTTTATCCATAGGAGCCAAAGAGGAGGCAGGCAGGTGGGAAATTGTTTTCCGTTGTCTGGAGAATGTGGTAAAAGTAAGACTGTGCATCATCCTTAGCCTGGGACCTGTATTAGGCCTTTATGAGACTGTCAGGAAATCTGCAAATGAATGGGTTTGGTGGAACTGGGATACAGGATGTGGGAGGCTCTGTGCAGTGTGATAATCCTGAAGGGAGGTGGGAAATTCTTCAGCCTTGTTTAGGTCTTGTTCCTAGCGCTGAGCGTGGCAGCTTCCCAATAGCGGGATGGTTCACTGGGAAGGAGCTAAGTTCAGCCAGATGAGTATAGAATTCGGAGCTAGccagactgaggaaaggtccaaagttgaccagattgtcccttccagcagctcaggcacagggtgctgattcttttaaaagttggtaatccaaacaggatgggctataattcgaactgcagtgcctgggagaggctaatcagtaggttctaggtctctccatctctgtgatatactgggtaggagtttctgcactctttgttcttgacctcttgtccttgcacgcagcagagttctgcacgagtagatggagtccgcctctcattttcctgttgtcctttgcagcccctcccacctttacagaaacacccccccagtacatcgaggccaaggaaggtggaagtattaccatgacgtgtactgctttcgggaaccctaagcccatcgtcacctggctcaaggaagggaccctcctcggtgctagtgcaaagtatcaggtgagtcagctctcacctgagtgccacagccacgcttcctgaccctctctccctgcccatctcccatggaatgcagctggccaccgaaggtatgcgtacacggcccagggtggaggcaggcaacctcatggcctgacttgactacagacattctgctgcctcttgtagtgaggcccttgtcctccctgcccctgtacgtgagTCTTTGAGGCTATTTCCAGCTTCTCAGAGCTCATCTTCTCACTTAAGAGGCATCCTGGTGAGGCTCACAGGTTGATCTTGAGTTCTTTCTGTGTCCCTAGGTGAGTGACGGTAGCCTAACGGTGACGTCAGTCAGTCGGGAGGACAGAGGCGCCTATACGTGTCGAGCATATAGCATCCAGGGTGAGGCTGTGCACACAACCCATCTGCTTGTTCAAGGTAGGGGGACTTTTTTAGCCTCTTTATCCCTGGAACTCTGCAGGAGGCTTCCAGGCCTTTTGAGTCATGGAGAATGTTTTTCTCATGGCACAGTCTGCCCACTTGGGGGTGGTAGAGGAGCTGAGTGGGACTGTCTTCACGTGTGTCACTTACCATGTCTCTCTCACCTTGGTCAGCTGGGAAAAGAGCATCTCATGGTGTGGCAGGGAGACAGGCATGCTGAGCCATTTTTGATATTCCGTTGAGAGAGCATTTCTACCATGTCTCCACCTTCCTCAGAGAGGAAACACCCTTGGGTCATCTGTCATTCAGTGATGACATCTATT

This mutation is a 636 bp deletion beginning at Chromosome 9 positive strand position 27,317,073 bp and ending after 27,317,708 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37758 CAC TGG GAA GGA GCT AAG TTC A Common A
37759 CTG CTG GAA GGG ACA ATC TG Wild type Reverse A
37760 TCT TAA GTG AGA AGA TGA GCT CTG A Mutant Reverse A
37761 Fluorophore-1 AGA CTG AGG AAA GGT CCA AAG TT Quencher-1 WT Probe
37762 Fluorophore-2 CTT TGA GGC TAT TTC CAG CTT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37758 0.40 uM
37759 0.40 uM
37760 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.