Protocol 34649: Probe Assay - Klhl24<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

>chr16:20114348+20114479 132bp AAATCCTTGTAGATATTTTCTGTGG AAGCCCTAACGCCATGACA

Mut= 105 bp

Wt= 132 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TTCCAGGGTATCCAGTATCCAGTCCCATTCTTTGACCTCCGTAGAAACCAAGCTCATGGGTGGTACACATACATACATGTAGGCAAAACACTCATACATGTAAACCTAAAACACTTTAAAAGAAAAATCCTTGTAGATATTTTCTGTGGCATAATGATGCCACTAATGAATCATTGCAAAGTAAATCACTGGCAGcctgtactgttcagcatatctgcaaggacaaatttaattgcctgtcatggcgttagggcttgattatattttaacagtcttgattatattttaatataccacttaactattatttgtgcattcttaacaataactattttaggtctactggctattcagaggtgatagttgtggttggaggctgtgaacgagttggaggatttaatcttccatacactgagtgctatgatccagtaacaggagaatggaagtccttggccaagcttccagaatttaccaaatccgagtatgcagtctgtgcactgaggaatgatattcttgtttcaggtaaatacaaattattatagtagctacaacttttgattgggaatcagattggctgtttttaaatgataatgacttactgagtgtaaaatgaatatttcagataaacaagtttatacaacaaaactgaaaagactttttgcccttgaaagcAAGAATACTTACAAAAGTTCTACCAAAAATTGGTTTGGTCATAATATTTTAAATGCTTTTATGTACATTTAGAGTATATGTATTTTAACTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTATATATGTGTGTGCCCAGAAGAACATAGAAGTG

This mutation is a 476 bp deletion beginning at Chromosome 16 position 20,114,420 bp and ending after 20,114,895 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44142 AAA TCC TTG TAG ATA TTT TCT GTG G Common A
44143 AAG CCC TAA CGC CAT GAC A Wild type Reverse A
44144 CCA ATT TTT GGT AGA ACT TTT GTA AG Mutant Reverse A
44145 Fluorophore-1 CTG TTC AGC ATA TCT GCA AGG A Quencher-1 WT Probe
44146 Fluorophore-2 AAA GTA AAT CAC TGG CAG CAA GAA TA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44142 0.40 uM
44143 0.40 uM
44144 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.