Protocol 34479: Probe Assay - Dnajb4<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

>chr3:152186249-152186338 90bp AGCGCTGGCTTTTATCCAGT TGTCATCCAGAAGAGAGCTATAA

Mut= 92 bp

Wt= 90 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

TTAAAACTATTAAATTTGTAATACTTAAGTATGTTAGAACAGGAGTTTTTGAGGTAAGGGAGTCTCAATTTCCTTTTATTTTTCATATTTATTGTGAAACAAAAGATTCAAGAAGAAATTATTAAAGCAAAGAATTTGGAGGAATGATTTAAAAGCTAAAACGTTAACataggatggatcttcagaaactattctgtttaattctaatgatgtttcaggccgtaagtgataagtcagagtttattagcagggcaatctgtaagtggtcagctgtctagactgccggcccttttcctaatcttgcttatgtcttcaccaggcttgaagggaggcgctggaggtactgatggacaaggaggtaccttcaggtacactttccacggagatcctcatgccacatttgcagcctttttcggaggttccaacccttttgaaatcttctttgggagacggatgggtggaggtcgagattctgaagaaatggaaatagatggagatccctttagtgcctttggattcagcatgaatggatatccaagagacaggaattctgtgggaccctcccgtctcaaacAAGACCctcccattattcatgaacttaaagtatcgcttgaagaaatatatagtggctgcaccaaacggatgaagatttctcgaaaaagattaaatcctgacggaaggagttacagatctgaggacaaaattctcaccattgagatcaaaaaagggtggaaagaaggcaccaaaatcacttttccgagggaaggcgatgaaacaccaaatagtatcccagcagacattgtttttgttattaaagacaaagaacatcccaagtttaaaagagacggatcaaatatagtttatactgctaaaattagtttacgggaggtaagtcagtaagtcagccctgagactctaagacggagagatgactgctgctgattgactagggggctgtctgggcagcgctggcttttatccagtgctctgcagacttattcctcatggtaaCTCTCTAGGAAGTGGTGCTATTATAGCTCTCTTCTGGATGACATCATAGTTTCTAAATTTATTTACTGATGTTAACTATTTAAATGTCGGTTTTCAAGTACAAGTTTGGTTAGCTACTTTATGGCCAGTGTTAGGTTAACTCTAGTTTTAGGTTGTGGTCTGTGAGATCCGCA

This mutation is n 837 bp deletion beginning at Chromosome 3 position 152,186,292 bp and ending after 152,187,134 bp (GRCm38/mm10). Also, after the deletion of 381 bp of sequence there is a 6 bp [GGTCTT] endogenous retention followed by an additional 405 deleted bp.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43718 AGC GCT GGC TTT TAT CCA GT Wild type Forward A
43719 TGT CAT CCA GAA GAG AGC TAT AA Common A
43720 AGC AAA GAA TTT GGA GGA ATG A Mutant Forward A
43721 Fluorophore-1 CTG CAG ACT TAT TCC TCA TGG TAA Quencher-1 WT Probe
43722 Fluorophore-2 AGC TAA AAC GTT AAC AAG ACC CTC T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43718 0.40 uM
43719 0.40 uM
43720 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.