Protocol 34644: Probe Assay - Klhl40<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

>chr9:121780106+121780213 108bp CCTTAGGGGACAGGATGGTC TTTCAGGTCTTCACCCAATG

Mut= 106 bp

Wt= 108 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 ACAGGCCTCACAGCTGGAGGAGGAGAGGGGCAGCTCTCCCAAGCTGGCTGGGGGAAGTTTCAGAGGTTGCATAGTATCAAGTGTCCATCCACACAATGCAGCTGTTGGACAACTCTCCAAGGTGGGCTCTTTGTGCCTATAACCACTCGGTTAGgcagataaaaagggtggcatggaaggctatagattttaagggacttgggggggggctggcgggggggggaagccttgaacagttggtcaccaaagatccaaagccacagggggacctccctctcagagggggaaacttggctgatctgggccaggaggtgagcacaggaggtgagcacaggaggctcatggtggctgacccaaggatactgcctgcccacagtttgaccacttggactctgagtggctggggatgccgcctctcccttcacctcgctgcctcttcggcctgggggaggctctcaacgccatctacgtggtcggcggccgggaactcaaggacagcgaagacagcctggactcagtcctgtgctacgacaggctgtgagtgggcaggacggggccaccttaggggacaggatggtctggggcaggggccctagggtggtgctgggaccacagcagcaaccagGAGGCCTGCAAGGGTGCACAGCCATTGGGTGAAGACCTGAAAATAATCCTTGAGCAGAGGGGGAGATGGGCTGGCTCAAAGGCAGGAGAGCTCAGGGGAGGGGTTCCAGGAGTGTATTGAATGCAGTCTGCATGTGTCTTGTGGGTATGAGTGCAGAGA

This mutation is a 471 bp deletion beginning at Chromosome 9 position 121,779,701 bp and ending after 121,780,171 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44116 CCT TAG GGG ACA GGA TGG TC Wild type Forward A
44117 TTT CAG GTC TTC ACC CAA TG Common A
44118 ACA CAA TGC AGC TGT TGG AC Mutant Forward A
44119 Fluorophore-1 CTG GGA CCA CAG CAG CA Quencher-1 WT Probe
44120 Fluorophore-2 CCA CTC GGT TAG GAG GCC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44116 0.40 uM
44117 0.40 uM
44118 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.