Protocol 37691: Probe Assay - Kbtbd13<em1(IMPC)J> Alternate 1
Version 2.0

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

>chr9:65390272-65390429 158bp TGTATCCACGTTGGTCTATGC AAGCTCACAACTCTGGCAGT

Mut = 164 bp

Wt = 158 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GGTATTTATAACCCCCAGGGAAGTTTTTTTTTCCCCTCCCCGCCCCGGGGTAGCCCCAGAGTCAGTGGCCCCTGGCAAATCTAGGCCCCCCAGCGGCAGCAGCCTCCACCCCTCTGGCTCATCATGCCCCCAGGCCccgaggtcccagtgcaggtgtgggtggacggtcaactcttccaggcggagcagtcgctgctggtggagcactgcggcttcttccgcgggcttttccgctctggcatgcgggaggcgcgcgccgcggaggtgcgcctgggcgcgctgagcgcctccggcttccgcacggcgctgcgggtgctgcgcggagagcggccggcgctggcggccgaggacgagttgctgcaggcggtggagtgcgccgcgttcctgcaggcgcccgcgctcgcgcgcttcctggagcacagcgtcacgtcggacaactgctcgcttttgtgcgacgcggccgccgccttcggcctgcgcgacgtgctccacagcgctgcgctcttcatccgcgacgggtcgcacgagctggtggcacaactggagctgcccgaggcccgagcctacgtggcggcgctgcgacccagcacctacgtggcggtgagcacgcacacgcccacaccgggcttcctagaggatgcgtcgcgcaccatgtgcttcctggacgaggaagaggacgcatggcgcacgctggccgcgctgcccctggaggccagcacactgctggcgggtgtggccaccctgggcaacaagctctacatcgtgggtggcgtgtgtggtgccagcaaggaagtagtggagctaggcttctgttacgacccagagggtggcacctggtgcgaattccccagccctcaccaaccgcgctatgatatggcgctggctggcttcgaaggccgcctctatgccattggtggcgaattccagaggacacccatgagctccgtggagtgctatgatcccgccacaggttgctggagcttcgtggcggacttgccacagccggctacaggggttccctgtgcccaggcacgtggccgcctcttcgtgtgcctgtggaggccggcagacatcactgccgttgtcgagtatgtggtgcaaatggacaagtggctgccagtggctgaactgtgtcgttcacagagctacggtcactttatggtggcccatcgcgatagtctctatgtagtacgaaatggaccttccgatgatttcctgcactgtgccatcgattgcctcaacctggtcacaggccagtggacatcgctacctggccaatttgtcaacagcaagggagcactcttcacgtctgtagtgcgtggggacaccgtttatactgtcaaccgtgtatccacgttggtctatgccattgaagatggcacctggcggctgctcagggaaaaggctggctttcctcggcctggctctttacagacctttcttctgcggctgcctcctggcactactggacctgtgGCCACCGCACTGCCAGAGTTGTGAGCTTGGAATGACTTCAGAAGATAACACAAGTCTCCATGAACCTTGGCGGTGTCCTCATGAGCCTTGACTGTGTGATTGGGGAGCTCTTGGGAACTCCTTTTTCCCTATTA

This mutation is a 1340 bp deletion beginning at Chromosome 9 position 65,390,300 bp and ending after 65,391,639 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50922 AAG CTC ACA ACT CTG GCA GT Common A
50923 TGT ATC CAC GTT GGT CTA TGC Wild type Forward A
50924 GGT ATT TAT AAC CCC CAG GGA AG Mutant Forward A
50925 Fluorophore-1 CTC AGG GAA AAG GCT GGC TTT Quencher-1 WT Probe
51949 Fluorophore-2 CCC CTG GCA AAT CTA GGC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50922 0.40 uM
50923 0.40 uM
50924 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.