Protocol 36191: Probe Assay - Dydc2<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

>chr14:41062722-41062847 126bp GGGTGAGTGCGTGTGTATTTC TTGTAAGAAAGGGTGCATGGT

Mut= 110 bp

Wt= 126 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CCATCAGCAGAATGTCATGATTCAGATCTTAAAGATTCCCCCAAATGGTCACATGCTAAAGGCTTGTTTGTGGATGTGGCAATACTGGGAAATGACAAGAGTCCATAAGACTCCCCAGGAGCCTAGTGGAAGGAAGTAATGTCACTGGGTCACCATGCAGGAGCTCCCACCCCTCTCTTTTTCTGTGTGTGTGTGAGTGGGTGGGTGTGGGTGAGTGCGTGTGTATTTCGTGGGAGGGgttaagaatgtatgtccctcccttcttttttcctccttctttccttccattcttttcctaaagcgagttacttttaccatgcaccctttcttacaatgtttggcctcactataagccaagaatgatggaccaaaggaccataaagccaagcctttccctcttcagttgaagagatcaagcatttttgtcacagtaagggtgatccagaaatagttcttattcttcctggaacgaatctgacttgttagaatggatctactttgaaggttagattgcgcttgtataatagagcatttgatttggtgtgtcctaggttctcagcatggatactgcctacctgaagaactgctttggaaccggcctgacccaggcactggcagaggtggcaagagtgcggcccagtgacccaatagagtacctggctcactggctttaccattataggagtataactgtagctgaggaaaaggtgtgttgtgtcctgggcctgcttaggtagggtgcagcttgctcagaacaacttgaaatccaagttatagtcctgagttggccaggagagtttttggctcatctcccttctgtggattgctttgtcccagatgtctctgaagtatgcaggactcgggggggtaaggaggaaatctagcttaatgggttgtagaattgggttggcagctactggctagctaatgcttgcaatagtaagttcagtaggcctcattggcccaggcctatctcacatgagggagggctcctttctaagggatgtgacctgatttgtctctgtcatattaccctagagaaggcaagaggagctacagcttaaggaagcacgtgacagaagcgctgaggaagcaaaaacaacagaaatgctgaaagaagaaggctatcagattcagcagaagtgtgaaaagtgtcaccaggtaggaaggaagaatggaaaattcaactcagcctctatcagccagggGTTTCAGGAAATCCTCAAGATGCCCAGCACAGGGTAAAACACACTAAAGCTAGCCTGAGCCTGCTTGTCCCTCATAGCCACATATCAAGCATGTCCTCTATGATGCAATACAGATGTCAGAATCCAACACCAAACAGTTGAAAGCAAGTCCGCCTTCTTCAGATTAGCAAGCAGAACTTTAGTATCCCGAGCTTATCCCCGTCCCGCGGGAAATAAACACAGGACACACCAGATTCTTCCACGGTTAACTT

This mutation is a 970 bp deletion beginning at Chromosome 14 position 41,061,848 bp and ending after 41,062,817 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48078 GGG TGA GTG CGT GTG TAT TTC Common A
48079 TTG TAA GAA AGG GTG CAT GGT Wild type Reverse A
48080 TGG CTA TGA GGG ACA AGC AG Mutant Reverse A
48081 Fluorophore-1 AAG AAT GTA TGT CCC TCC CTT CTT Quencher-1 WT Probe
48082 Fluorophore-2 CCA GCA CAG GGT AAA ACA CAC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48078 0.40 uM
48079 0.40 uM
48080 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.