Protocol 43934: Probe Assay - Krtap1-3<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

>chr11:99591312-99591424 113bp GCAGCCAGCCAGGGATTAT CAGGCCATGGTGTCAGAAGT

Mut= 118 bp

Wt= 113 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

GAAGATTATAGTGAATATGGGTGATCCAGGCTCATGTTTCCAATGAACGCTAATGACTACTTCACTGTAGTCAAGAAGTCTAATTGAACACCAAATAAAATCAGCTTTTTACGGGCTTTCAGAAGTCTGAACAAGTAACTCAGAAACAAACAATTATCATTTAGAGAAAAACTACATAAACATAAACAAGGAAGACACTTTGGAAATTAGGGAAACAGCAGCCAGCCAGGGATTATAaaaagactggagtggcactcaccacacaaattcaaaaaacttctctaagcaaacaaaccttcagcacagatcaacttctgacaccatggcctgctgcgctactagcttctgtggctttcccacatgttccactggtggcacctgtggctccagctgctgccagcccagctgctgccagcccagctgctgccagcctagctgctcccagtccagctgctgccagcccagctgctgccagtccagctgttgccagcccagctgctgccaatccagttgctgccaaccttgctgctcccagaccagctgttgccagcccagctgctgccagcccagctgctgtggaactggcagtggccaggagggtggcagtggagccgtgagctgccgtgttagatggtgccgccctgactgccgcgtggagggcacctgcctgcccccctgctgtgtggtaagctgcacacccccaacctgctgccagctgcaccatgcccaggcctcctgctgccgtccatcctattgtggacagtcctgctgccgcccagcctgctgctgctactgctgcccacccagctgctctgagtctaactgctgtgagcctacctgttaagacccaggttgctgattcccaaaggatcttgaattttgatatggcttgtgtttggggcacaaatacttggccttactaaacatttttttccatctaatactggaaaggctttgatatcctctatgaaaataataaaatacaaacttgatccccaaacaagaaaatcaatttaccttgggattgtccctattttccaaggcttggtgctgtcaaggccatggaagattcttctacacgaagatagttttcattcttcttccattgttctgtaaattcctcttataaatgaaggctaatgtatccaaaataataaattatatctgttggcatatcaaaacttgcttctttttatttagtgtgcaaataatagatacttcatggagatccatctaaaagatttctgagtttgattctatacacttatctattttgtgtggggagtggggcatgtaagtagagatcagtctctccttccaccttatgattctgaggactgaactccagtcatcgggcttagaggcaagtgcctttacccactgagctgccttgtcagccctgtttttttatattcttcttatacctgttaggaaatgggatgaattccaacagtggttagaccccaaaaaagttgtagcattgccctggtcttgttttattttgctgccgtcaataaacattgtgtccaaaaacatggggagcaaagaggttgtttggcTTATACTTCCAGAACACAGTTCATCACTGAGGAAAGTCAAGGCAGAAACTCAGAGTCAGGAACTTAGTGGCTTGTTTTCTAGCTCACTTGTGGGATCAATTTTAGCTAGCTCTCTCATAAGGCCAGCCCCTCCTCAATCAATTAATAATCAACATAGTCTCTTGTAGGGATAGAGACAGTCACTTGGTTGAGGGTCCCTTTCGGGCTATCACAGGCTGTGTCT

 

This mutation is a 1322 bp deletion beginning at Chromosome 11 position 99,590,083 bp and ending after 99,591,404 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64388 GCA GCC AGC CAG GGA TTA T Common A
64389 CAG GCC ATG GTG TCA GAA GT Wild type Reverse A
64390 TGA TCC CAC AAG TGA GCT AGA A Mutant Reverse A
64391 Fluorophore-1 CAA ACA AAC CTT CAG CAC AGA T Quencher-1 WT Probe
64392 Fluorophore-2 AAC TCA GAG TCA GGA ACT TAG TGG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
64388 0.50 uM
64389 0.50 uM
64390 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
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.