Protocol 35704: Probe Assay - Klb<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

>chr5:65375590+65375751 162bp ACTTTTGTAATGTGCTGTGTCC CCTGCCACTCAAAGCCATC

 

Mut= 164 bp

Wt= 162 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 ACTTGGTCCCCTTGTACCATGCAGGCCTCTGACTCAACTTTGTCAAGCTTCATAGTAGGTGCCTTTACAGATGCAGCTATTTTGCTGGCCCCAAATCAATAATTTGTGGTTTGATATAAAATGCTTGATATTTAGGAAAAGAGTTACTTTTGTAATGTGCTGTGTCCTGgcactttgtaaacgaactagaatgttttagtcctctgaaaatcgctgcttccttttctctgtgcagcaataaaatttgatgaaatccgcgtgtttggttatacggcctggactctcctggatggctttgagtggcaggatgcctatacgacccgacgagggctgttttatgtggactttaacagtgagcagaaagagaggaaacccaagtcctcggctcattactacaagcagatcatacaagacaacggcttccctttgaaagagtccacgccagacatgaagggtcggttcccctgtgatttctcttggggagtcactgagtctgttcttaaggtaagtggctacagctgaattaaccacgcaccagGGGAGATAGTATACAGTATGGTTTTTTGTTTTGTTTTGTTTTACAGTATGTTTTTACAAGTGTAAAATACATGTAAGGATATCTTAGTCATTGTTCTGTTGCTGTGAAGAAACATCATGACCACAGCAAGTCTGGGGTCTTCAATTGGGGCTGGTTCACAACTCATCAGCATGGTGAGAA

This mutation is a 369 bp deletion beginning at Chromosome 5 position 65,375,614 bp and ending after 65,375,982 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46801 ACT TTT GTA ATG TGC TGT GTC C Common A
46802 CCT GCC ACT CAA AGC CAT C Wild type Reverse A
46803 AGA CCC CAG ACT TGC TGT G Mutant Reverse A
46804 Fluorophore-1 AGT CCT CTG AAA ATC GCT GCT TC Quencher-1 WT Probe
46805 Fluorophore-2 AGT CAT TGT TCT GTT GCT GTG AAG AAA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46801 0.40 uM
46802 0.40 uM
46803 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.