Protocol 34648: Probe Assay - Klhl22<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

>chr16:17777180+17777279 100bp TTGTGGGTTATGTGGGCTGT TACCCCAGAAAAGCTGTACCA

Mut= 101 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CAGGTTTTAAACTAAGCCTACAGAAAGTTTCAGTTTTCTGTCAGAAGAGGTTGGGGTCGGTTGAGGAAAGATTCCACTTTGCTTCTCCCAGTCCCAGAAGGCCAGCTACAGGGACAGCCAGCTCTGTATGCCTTACTTCTGCTGTCATCCTGTCTTTAATATTCAGATGCAGTCCTCCAGCTAAGACttgagtgggtatccttctttttacagataccagaaattatccacttctgctgtgatttcctcatgtcatgggtagatgaggagaatatccttgatgtctaccggctagcagacctctttgacttgaaccatctgacccaacagctagatacctacatcctcaaaaactttgtggccttctccagaactgacaagtaccgccagcttcccttggagaaggtctactctctcctcagcagcaatcgcctggaggtttcctgtgagactgaagtgtatgaaggggccctgctctaccattactccctagagcaggtgcaggctgaccagatctcactgaatgagccccccaagctcctagagactgtgcgctttcccctgatggaagctgaggtccttcagcggctgcatgacaagcttggtcctagcccactgagggacacagtggccagtgcccttatgtaccaccggaatgagatcctgcagcccagccttcagggcccacagacagagcttcgctcggacttccagtgtgttgtgggctttgggggtattcactccacaccgtctacaatcctcagtgaccaggccaagtacctgaatcctctactgggagagtggaaacacttcactgcctctctggcgccccgaatgtccaatcagggcattgcagtgctcaacaacttcgtgtacttaattggaggggacaataatgtccaaggatttcgagctgagtcccgatgctggaggtacgaaagggcacttaccatcattgcctttattgttagacaactccatggtgcaggaattgtgggttatgtgggctgttcagggtacacgttatcaagggcacatctctacagacCTGCATCTGCCTTTTATATGAATGGTACAGCTTTTCTGGGGTATACCTGTGTCATGTAGTGAAGTCCAGATGAATCCAGACAAAGTGGACCACAGTGAAATAAGGAAAAGGGCATCAGAATTGCTTTCTTGCCTTTTGACCCTGCTATCCAGAAA

This mutation is an 861 bp deletion beginning at Chromosome 16 position 17,776,376 bp and ending after 17,777,236 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44136 TTG TGG GTT ATG TGG GCT GT Wild type Forward A
44137 TAC CCC AGA AAA GCT GTA CCA Common A
44138 GCC TTA CTT CTG CTG TCA TCC Mutant Forward A
44139 Fluorophore-1 CGT TAT CAA GGG CAC ATC TCT AC Quencher-1 WT Probe
44140 Fluorophore-2 AGT CCT CCA GCT AAG ACC TGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44136 0.40 uM
44137 0.40 uM
44138 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.