Protocol 41600: Probe Assay - Atxn7l1<em1(IMPC)J> Alternate 1
Version 2.0

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

Mut = 124 bp

Wt = 118 bp

Fam = Mut

Hex = Wt

>chr12:33342244+33342361 118bp AGTCCCATCTGTAACCAGTGA GCACCATCTCCCAGGTACTC

Sequence

Wt Sequence (deletions in lower case):

CACCGTGCCAGCCTCTCACCCCCTGTTGGTCCTTTACCCGGCACAGATCTCCCTCCCATCAAAACGGGGAATCACGCTGCTCGCTTACAGCAGGAGCATAAATAAGTGATAAGGAAGTGTTGTGATAACGATAAgcgggggtgtagctcctcggtggaccagatctatcaaagaaggcggtggtgcatgtgtgttcctttatttttcagcgttccagttcccgtagtcagtttagagaaaattcctaacctagtgaaggcagatggtgccaatgtcaaaatgaactctacaaccactactgcagccacttcctcctcctctgccgtctccactccgcctttaattaagcccaccttgatgtccaagtcagtgccaccttcaccagagaagatcttaaatggcaaggggattctgtcagccaccatagacaagaagcacccaaacggcaccaaaaacagcaacaagccttaccggagactttcaggtatgtgttcctgtggttagagtctggctcacggtaagatgacaactgagtcccatctgtaaccagtgatgtgttcgtagaggcaaacagctgtttggtttgaacagcgatctggagtgtagctgcaaGTAGGACCGGCACCCCGAGAGTACCTGGGAGATGGTGCTCTCATCCCCCAGTGCAGATAAGGTCCCCAGCCAAGTTTTGA

This mutation is a 478 bp deletion beginning at Chromosome 12 position 33,391,845 bp and ending after 33,392,322 bp (GRCm39/mm39).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
57967 GCA CCA TCT CCC AGG TAC TC Common A
57968 AGT CCC ATC TGT AAC CAG TGA Wild type Forward A
57970 Fluorophore-1 ACA GCG ATC TGG AGT GTA GCT G Quencher-1 WT Probe
58737 CTC CCT CCC ATC AAA ACG Mutant Forward A
58738 Fluorophore-2 CAC GCT GCT CGC TTA CAG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
57967 0.40 uM
57968 0.40 uM
58737 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.