Protocol 35237: Probe Assay - Dok4<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

>chr8:94867548-94867702 155bp ACACTCACCTTGGCCATTTG CAACAATGCTGCCCTGAAGT

Mut= 146 bp

Wt= 155 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GGATAGGAAGCATTCCACAAATGTTCCTTCCATTACACAGAGCATCATAGAACAGCCCGATAGAGATGAACATCAACACTCACCTTGGCCATTTGGTTAGGGCTGCCCCTCTGTTGGCTGCTATCCTGTAGCTACCCTACCATCCtaaggttttctctaagctaggatccagggaagggtgtagtattgctccctcaggaggctggggtaacttcagggcagcattgttggggatcacggtccagcaacaatccgatcattagcttcttgacccacagatttaccggaggtgctggctggtgttccggaaatcttccagcaaggggccccagcggctggagaagtatcctgatgagaagtccgtgtgcctccgaggctgccccaaggttaggggcctgggtcctagatcccccaacctgtgagcaaagatgacttgctgagtcctgaggtctgagagtaccttcctggggaacccagctaaggaggtaaggtgaagtgccccggcctggctaaatctcttgggatatctttccaggtgactgagattagcaacgtcaagtgtgtcacacggctccccaaggagaccaagaggcaggcggtggccatcatattcacagacgactccgctcgcaccttcacttgtgactcaggtaaggccccaggactgcaggctccatctgctctcctcgggctggtcagtcagtggtctaggcagtgagtaccagagcaaggccaacccttgcagaaagagtgggagagctgtgcccttaGTGGAGTAGGTCGCAGACAGAGGTGGGCAAGGCAGGCAGCAAGGCCTTCCTGCATGGTGCAATGAGTCAACCCAGGGCAGATTAGGTGGGCTGCTGGGGCCTCTGAGCTGGCAGAACTATGAAGTGCTATTTTTTTCTAGAGCTGGAGGCAGAAGAGTGGTA

This mutation is a 625 bp deletion beginning at Chromosome 8 position 94,867,008 bp and ending after 94,867,632 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45585 ACA CTC ACC TTG GCC ATT TG Common A
45586 CAA CAA TGC TGC CCT GAA GT Wild type Reverse A
45587 CCT GGG TTG ACT CAT TGC AC Mutant Reverse A
45588 Fluorophore-1 AGC TAG GAT CCA GGG AAG GG Quencher-1 WT Probe
45589 Fluorophore-2 CCA TCC GTG GAG TAG GTC G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45585 0.40 uM
45586 0.40 uM
45587 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.