Protocol 34639: Probe Assay - Klhl32<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

>chr4:24792818-24792933 116bp AGGGCAAAATTGAGAACAGG GCCCTGTTTATGCCTTTCA

Mut= 111 bp

Wt= 116 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 AGCAAAATCAAAACCAAAATAAAAAACCCCAGGGAGCTACCAAGAGAAAAAGTCCCAGGATAGGGCAGAGGAACAAATTAGAGTAAAGGAAAACAAAGAAACAAAAAACAGAGAGGGCAAAATTGAGAACAGGAAGGTCAGACTCACAGGGCACTAGAAGTAGCCTCCCCAAAGCccaacgggtgagagagccccaggaagctgagccggtgaaaggcataaacagggcttcattactctcctttgtagtattcaagaaatgctgacgggccagaggctgtgccactcggaaactcacaatgacacggtcctggcggcgctgaatcagcaaagaagtgatggcattctctgcgacgtgaccctgatcgccgaggaacagaaattccacgctcacaaggcggtcctagcagcctgcagtgactacttccgggtaagtgaaggtggatgtgtttgtcttaggatgtcagtgcacagaggacagcctgatctttcctaggccgtgagccaactacatggcGGAAGGCAGATTTGTAAGTGTTGAGAACCTGTGTACCTTCctggacagaacTGAGTCCAGGACCCTGGTGGGACATTCACTGGACTGGGCTGGGTGGCTGCAATGAGAAGCAGACCGTTGCATCTGACTAATACATTTAACAAGCACTGAATGGTGTGGCAGTGACATGCATGGCATATGAAGATATGGTTCTTTTTTCCTAGAACTTGAAATTTGATTTCCAGCTGTGAGTG

This mutation is a 342 bp deletion beginning at Chromosome 4 position 24,792,530 bp and ending after 24,792,871 bp (GRCm38/mm10). There is an additional 11 bp [CTGGACAGAAC] del 40 bp after the del site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44093 AGG GCA AAA TTG AGA ACA GG Common A
44094 GCC CTG TTT ATG CCT TTC A Wild type Reverse A
44095 CTG GAC TCA GAA GGT ACA CAG G Mutant Reverse A
44096 Fluorophore-1 CGG GTG AGA GAG CCC C Quencher-1 WT Probe
44097 Fluorophore-2 CAA AGC GGA AGG CAG ATT T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44093 0.40 uM
44094 0.40 uM
44095 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.