Protocol 34643: Probe Assay - Vps26c<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

>chr16:94501187-94501291 105bp GCATTCAGCTTCTTGATGACTC GCTCACAGTTGACTCTGGATG

Mut= 100 bp

Wt= 105 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GGGAACACTTTAAAACGGCCTTTTTCTTTTTCTTCTTTTGTGACCTGGGGTTGAGGGGGTGGCTGTCCTTAAGCACCCTTCTAACCCTGAGGCTTTTTACATTTCATGTCTTTCGGCCTGAGAGAGCTTTGTCAGCCAGTGTAGGTTGGGAGCTGATTCGGAGTGAGCTGTGATgtccgtttgtccctaatatgtgtacagggtggttgatgagtcagtctggtggccactgaaagatgaaatggctattattccttcttgttttagaaagaagtttccatggtttatagaacaattactcaacttggttttcagttcttatttgtaccagtgcaactggaataagtgacagccccctttccttctgtttctgatgtctttgtctctgttctccatagagagcttcacttcccaaattcttcattagaggacatctgaactccactaactgcgctatcacgcagcccctgacaggagagctggtggtggaacactcggatgctgctatccggagcatagagcttcagttggtccgggtggagacctgtggtaagacttctagagtatctaggtatctacacttgtactgtcagccgctaaaacaccccttcctgtactcttaacatggcagggcacatcacatcataacagagctattctgatggcccctgtggcctactcaccccaaaattcccacttgcagataatcactgaaatactaattaggtccccagcaccttaaatcttggaagcatggtaaaaccagtgtggtgatgtttgagaacatgcaaagtagaagagaaggtaactcccagtcctgaagaagaaattggagaagtgggtgaatgtgttgatcctggttggcacagtattgggcagtttggtgaccagtttgctttggtgtcatggtgtcctgaccccatgatgtttcctctttctccagggtgtgcagaaggctatgcacgagatgccacagagattcagaatattcaaatcgctgatggggacatttgtagaaacctttctgttcccctgtacatggtcttccccagactgttcacctgtccaacactggagaccaccaacttcaaagtgggtaagcagtccaggcctggcctgtggagtctaggaagggtctgggccacagggaagaagcgccttgcacgcaggctgcagctttttttttttttttttggtttttcgagacagggtttctctgtgtagccctggctgttctggaattcactttgtagtccaggctggcctcgaactcagaaatccgcctgcctctgcctcctgagtgctgggattaaaggcgtgcgccaccacgcctggcttagcattcagcttcttgatgactcaaaaaagggtaagaagctgcgtaagtagGTGTGTGTAGGCAGTAGAGCCTGCTGCAAGGCCCCATCCAGAGTCAACTGTGAGCCAAGCAGAAATACCGTGAGTCTATAGGAAAAGCCTTGACAGTTGTCAGGTAAGTAGTATGTGAGTAAATATCCAACAGGAAACAACTTAA

This mutation is a 1215 bp deletion beginning at Chromosome 16 position 94,501,242 bp and ending after 94,502,456 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44111 GCA TTC AGC TTC TTG ATG ACT C Wild type Forward A
44112 GCT CAC AGT TGA CTC TGG ATG Common A
44113 TGT CAG CCA GTG TAG GTT GG Mutant Forward A
44114 Fluorophore-1 AAA GGG TAA GAA GCT GCG TAA GT Quencher-1 WT Probe
44115 Fluorophore-2 CGG AGT GAG CTG TGA TGT GT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44111 0.40 uM
44112 0.40 uM
44113 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.