Protocol 33828: Probe Assay - Dhx38<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

>chr8:109562326-109562457 132bp AGCCGGAGAGACAGGTAAGG CCTGCCACCAGTATCATGGA

Mut= 134 bp

Wt= 132 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TGGGAGGAGAGTAAGGATGACCAGAAGGATGCCGAGGAGGAGAGCGAGGACCAGGCTGGCCGAAGTAGCCGGAGAGACAGGTAAGGAGAGAGGGTAAAAAGTGGTGGTCAGAAAATATTCGCCCAGgttcacctcagctcctgtcggacacctaagagctttacggttttattgtgattccatgatactggtggcaggtaggagctggtaaggatttgcggggaaggctgctaagaagtggacgtaagtgcttgggttctagccctgtccaggccttcagcaccagggagatgcagtggagaggacatactgcttgttggcatggtcggaggtagagggaaatgttttctgccatggagacccctttcttcactctccctgtttcaggcattaccgatctgctcgggtagagactccgtcccaccccgggggtgtgagtgaagagttttgggaacgcagtaggcagagagagcgggatcggagggagcacggggtctatgcctcatctaaagaagaaaaggatcggaagaaggagagatcccgggatcgagactatgaccgcaagagagacagaggtaacgtgcctggcacccctcctggggtccgttagcatcgaaccagcaagcctttctcttcctgtcacaaggacttttacagtaatagtcattaattggacaacttggtttgtatagtgtgtgccccggtgGTTTTGACCCTTTGAGACTAGGATTAGGAAGAAGTTTAAGGAACGATACCTCTCTTGTAGGACAGCAACTTTGCTTGAGTGTGTGGATCTTGTTTTATTGTGAAAATTGGTGATAAAGGAAATGATGGGTGATCCATTGAAGAAGCCAGTATTTACACTA

This mutation is a 580 bp deletion beginning at Chromosome 8 position 109,561,818 bp and ending after 109,562,397 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42282 AGC CGG AGA GAC AGG TAA GG Common A
42283 CCT GCC ACC AGT ATC ATG GA Wild type Reverse A
42284 GCA AAG TTG CTG TCC TAC AAG A Mutant Reverse A
42285 Fluorophore-1 CCT CAG CTC CTG TCG GAC Quencher-1 WT Probe
42286 Fluorophore-2 CCC AGG TTT TGA CCC TTT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42282 0.40 uM
42283 0.40 uM
42284 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.