Protocol 44060: Probe Assay - Hs1bp3<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

>chr12:8323625+8323726 102bp TCTGGGAACTCCTGCCTTAG CAAGTGCTGTGCTATCCCACT

Mut= 84 bp

Wt= 102 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

AATTAAGATAGACGGACAGACAGACACACAGTTAACAGTTGTTAGGAGAGAGGCTTTTCTCACATAGTGTAACTGTTATAAGCACCCCATGCCCCTGTAAGTAGCCCCAGTTAATTCAGCTCGTTAAGCTACACATGGGTGGAATCCTACTGCTGTTATGCAGATAGCACCCTATCAACATAGAATGTCTCCCTGGAAAAAGTTTATAAAACACTTCTGGGAACTCCTGCCTTAGGCTTCGTCTGACGTTCCccatggccaggcagcagcggggtcccttgagcgtcatgtcacatagtgggatagcacagcacttggcgctctccagactctccacttaggtgtcaggaggttaatgcaatccctcagcctcagaacctggcttaggcttaggcaggtgggtgtggtgatgtcgccttgagagtagacagcaagctcttgttttccttttcttccacaggcaccagagccccaggggctacgggccttgccacccgagatccctctgtcttggatgacacagccagccagccaggggacagtgatgaggcttttgacttctttgagcagcaggatgaagtgcagccacccacactgggcctgagcagcaaggatgtggagaagtccttggtgggagaagaggaggaggaggaggaggaggaggaggtgctggatcccctgggtatcatgcggtaggtctccacttcttccgatgTCATTTGTTCAAGGTCTGGGCCATGGGGCTTTAACCTGGTGTGCTCAAGAGAGTTGGCTCAGCTCCGCCGCCTCAGTATCACCCGCCTTTGTGGCTGGGCTCAGAATCCCCCGCCCTCCCCAGTGATATCCCCCATCTATGGCTAACTCCACAGAGTTAACCCCACGATAGCTCGGTAGCACAGACTGCTGGGCCCAGTCTGCTGCCATTGGGACCCGGCTTCTCCTTGCAGAGTGGCAAGTGACTTGTTTGCCCCCAGAGCATGCTT

 

This mutation is a 463 bp deletion beginning at Chromosome 12 position 8,373,662 bp and ending after 8,374,124 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64685 TCT GGG AAC TCC TGC CTT AG Common A
64686 CAA GTG CTG TGC TAT CCC ACT Wild type Reverse A
64687 TGA GCA CAC CAG GTT AAA GC Mutant Reverse A
64688 Fluorophore-1 CCC TTG AGC GTC ATG TCA C Quencher-1 WT Probe
64689 Fluorophore-2 CTG ACG TTC CTC ATT TGT TCA AGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64685 0.40 uM
64686 0.40 uM
64687 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.