Protocol 34952: Probe Assay - Aco1<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

>chr4:40164527+40164617 91bp TGAGGATTGGCTTTCAAGAGA GTAAGCGTCCTGGGGATTAAG

Mut= 96 bp

Wt= 91 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CAGACATTCAAATTCTGAATGTCTGTTCACTGTACATAGTTTTCACCTTATAGACATTTTAAACTTGTCAATCACTTGGCCCTCTTTCCCTGTTAGAAGAATAATCCGATGTCATGTGTCAAGTTTATCTGAGGATTGGCTTTCAAGAGAGTTGTGAAAAGCAGGTGCCctgtggtttgaggttattaatgctttctaccttaatccccaggacgcttaccattttctatcagagttctcctggaggcagccgttcggaactgtgatgagtttctggtgaagaaaaatgacatcgagaatatcctgaattggaatgtcatgcaacataagaacatagaagtgccgtttaagccagcccgagtcatactgcaagactttacgtgagcctgacacctcacctcacctcacctctctctctctctctctctctctctctctctctctctctcgctcgctcgctcgctcgctcgctcgctcttccctgcccccccctgcctcccttgtcagcctcccaagaaagtttttactgtagcaaaaatgctttgtctctgagaaaccaggagataaaaggaaggcaaggggctcctggaacaagagaagcctgctctgtctctagtagtcaccccgcctcaccagtgacAGGGGTACCATCCTCAGGACTGATCACACTGGGAAGGGTAGAAGACAGCACGCAGGAGGGCTGTGTGGAGTCTCTTGTGCAGGCTGCTGTGCCTGCATACAGTTGTGCCTACCGACCTCAGGGATAGACGAGGTGCTTCGTGAGGCCTGGCTTGCACTCGGCTTGCA

This mutation is a 471 bp deletion beginning at Chromosome 4 position 40,164,567 bp and ending after 40,165,037 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44894 TGA GGA TTG GCT TTC AAG AGA Common A
44895 GTA AGC GTC CTG GGG ATT AAG Wild type Reverse A
44896 CCT GCG TGC TGT CTT CTA CC Mutant Reverse A
44897 Fluorophore-1 CTG TGG TTT GAG GTT ATT AAT GCT TT Quencher-1 WT Probe
44898 Fluorophore-2 AGG TGC CAG GGG TAC CAT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44894 0.40 uM
44895 0.40 uM
44896 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.