Protocol 34763: Probe Assay - Wdr74<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

Mutant=  85 bp

Wild Type = 88 bp

Sequence

Genomic sequence with deletion in lower case

AAGCAATCTAGAGCTTAAGCCTAGGGTGGCAGATGTGTGGAAGAACACAGACACAGTTGTGCAGAGCCTGGGAAACGGCTTGGGcttccagggaagaggtttatgttatcgttgtttgggttgggttgtttatttctgggggctgggggagggaaggtatgtatgttttgttgtttagtatctcatgtagccaggatggccttgaactcactatgtagctcagactgacgtggaattccaggttctctctttactccccacactggtagctgtgcaccataaaacctggcttatactttgtaaaatcccaatattctcttgcttgctttcagcacccttatcacatgtgtggattctgggatcctcagagtctggtgtgaaaatgacaaagaggcgtcctctgacccagtaaggtttacctgtcccggtggttgaaaggatgggagctagggtaggggtctgatggggaatttggtaaaggaaagattaagggctttgagactaactcatctctggttattttcagctcttggaactgaaggtgggtcctggggtgtgtaggatgcgccaagatcccacacatacacatgtggttgccacatgtgggaaagaaaatgctttgaaagtatgggacctacaggggtctgaggagcctgtattcagggccaaaaatgtgagtgagtgatgaagggttggggagatgctatgtttgattgaggttggggaagtgaaagccactgacatgacctagatggctttgggggtatggaggattggaggaaacaggtcaggcttatcaaactttgtgcccatatgtaccttccacaccaggtgcggaatgattggcttgatctccgagttcctatttgggaccaagatacacagttcctccctgggtcacagaaacttgtcacatgcacagggtaccaccaggtgaggctctttttactcccggattgaagtagcttgaattcagaaaaacaacaaaaaaagtgtctcctgatagagtaggtaggttgcatggtgtaactctgcctgggttatcaaaggagctcatactttcttagtcaacaaagCCCATGGCTGAGCCTACTACTTTCCCCTTACTGCATTCCAGGGTGGAATTCTGAACTCTGGGTTTCTATTTCTCCTTCCTAGAAG

This mutation is a 992 bp deletion beginning at Chromosome 19 position 8,737,406 bp and ending after 8,738,397 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44407 TGT GTG GAA GAA CAC AGA CAC A Mutant Forward A
44408 GCA GTA AGG GGA AAG TAG TAG GC Mutant Reverse A
44409 CGT CCT CTG ACC CAG TAA GG Wild type Forward A
44410 TCC TTT ACC AAA TTC CCC ATC Wild type Reverse A
44411 Fluorophore-1 AGC CTG GGA AAC GGC TTG G Quencher-1 MUT Probe
44412 Fluorophore-2 AGG ATG GGA GCT AGG GTA GG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44407 0.40 uM
44408 0.40 uM
44409 0.40 uM
44410 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.