Protocol 36172: Probe Assay - Dnajb14<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

>chr3:137885308+137885444 137bp GCTGGAAGTAGCACGCATTG CTGAGCACGCCCTCTACTTG

Mut= 138 bp

Wt= 137 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case; and insertions with carrots ^g^ (TTCATAGC & T insertions):

 GAGGGTTTTACTGTGTAGCTCTGGCTTTCCTGTAGTTCACAAAGATCTGTCTGTCCAGCTCTGACAAGCAACCTGTTACTTAAAGAGACATTATGAAGCCTGAACTGGTGGCTTGTGTTTGTAATCCCGGCACCGGGAAACTAAAGCAGAGTTGAGAGTTCAAAGCCGGATTGGGCCACGTAATTCTAGGTCAGCCCCAACTGCATGGTAAGGTCATGCCTAAACAAAGGGAAAAACATAACAACAATCTCCCCTCCCTAATAAAAAAGATAAAGGAGGACGAGGCCATGT^ttagtctctccagaagagctcatcttgtctgctttgtcttttttccggagccctttgtctctgtgtcctgatgtgagtaggttgaagtgtcctttatctctctttacacagcgttattggaaataattatgaaaaacggcagcaccgctggaagtagcacgcattgccgaaaacctcctgggagcagcgaccagagcaagcccagctgtggaaaggacggcacttctggtgctggggaaggcgggaaagtctataccaaagaccaagtagagggcgtgctcaggtaggtgtgaatgcgagtgtgtccgcaccatgatcgttgactgtgattggacccaatcgacgttataatgcctaaaataaaaatgtatttatatatagcacagtttaatagag^GGAAATGAAGG^AGTTACCTGGGTGCCCTGTCCTTCATTATTTTGAAGTCAGCCTGGAGGGAGTTAACTTTCTGGAAGTTTCCACTCATTTAAATTAGCTATAAGTCACAGTGAGTTTTTAAAATAATGATCTCAGCTTTGAGTCTATGCACTGTTTGGACTGTAGTATTGAATGGATTGCTTTGTTGTGATACGTAGTTGAAAATAGGAGAATTTCATATGCGGGAAGGCT

This mutation is a 396 bp deletion beginning at Chromosome 3 position 137,885,162 bp and ending after 137,885,557 bp (GRCm38/mm10). In addition, there is an 8 bp insertion (TTCATAGC) at the deletion site and a single bp insertion (T) 11 bp before the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48011 GCT GGA AGT AGC ACG CAT TG Wild type Forward A
48012 CTG AGC ACG CCC TCT ACT TG Wild type Reverse A
48013 TCT AGG TCA GCC CCA ACT GC Mutant Forward A
48014 CCC AGG TAA CTT CCT TCA TTT C Mutant Reverse A
48015 Fluorophore-1 CTC CTG GGA GCA GCG AC Quencher-1 WT Probe
48016 Fluorophore-2 CGA GGC CAT GTG CTA TGA A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48011 0.40 uM
48012 0.40 uM
48013 0.40 uM
48014 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.