Protocol 35019: Probe Assay - Atp8a2<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

>chr14:60046746-60046856 111bp TGCCCTCCTACAAAGGACAT CTGTACTTGGCCGTACTGGA

Mut= 111 bp

Wt= 111 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CCGAATGAACCTGAATACACACATGTTGCTCTTAGCATGTGGCTCCCAGATGGGAGTCACTGGTCTCGTCACCCCATGGGAAAGTGGAATGTCCATATCAAAGATTGAATTGCAAACACATTGGAAGTGTCCCTGGTCAGgtgtgccctcctacaaaggacatgcagttggatgtcactttgctgttatttaagggttacctctttaactaaaagtcgtacttgtctttgtacttccagtacggccaagtacagcgtgttgacatttctacctcgattcctgtatgagcagattaggagagctgctaacgccttcttcctcttcattgccttattacaggtaatggtttttaaggacttaaacattgcaagctcaattatgtgcaaaatgagattctttagtaagtgcctttattggaaaagtgttagaatatttacatagtgtccttcctcaaattatcactaggggggacagacctgatcttggtataaaacaaacgtgggagGGAAGTCTTTGAAAAAAGAAGTTACAAAAGGGGAGCAGAAACTGGTTCTTGTGGGTCCTACCTTGTCATGTTTACCTTTTCTTGTTATTTTTAAAATTATTAATGCACATTCATTATATTAGACATGATATTTCACTGTGACACTTCATACATATATACCATTCATG

This mutation is a 365 bp deletion beginning at Chromosome 14 position 60,046,495 bp and ending after 60,046,859 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45038 TGC CCT CCT ACA AAG GAC AT Wild type Forward A
45039 CTG TAC TTG GCC GTA CTG GA Wild type Reverse A
45040 CCA TGG GAA AGT GGA ATG TC Mutant Forward A
45041 CAG TTT CTG CTC CCC TTT TG Mutant Reverse A
45042 Fluorophore-1 CAG TTG GAT GTC ACT TTG CTG T Quencher-1 WT Probe
45043 Fluorophore-2 TGT CCC TGG TCA GGG AAG T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45038 0.40 uM
45039 0.40 uM
45040 0.40 uM
45041 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.