Protocol 39493: Probe Assay - Aco2<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

>chr15:81895045+81895152 108bp TGCAAACAAGCAGAACTTGG GAAGTAGCTTTGGGACTCAGC

Mut= 96 bp

Wt= 108 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

TCTTCAGCTTGCTACATAGCCAAGGATAATTTTAATTCCTGATCCCTGAGCCCTGCCTCCCATAATGCTGACATTCACTTGTTCTTCAAGTAAACTGCAAACAAGCAGAACTTGGGATCCTGCACACAGATGCTTCTGTGCCTGCTCACTGCCACACAtgtgagagggaaaagctgtgccaggctgagtcccaaagctacttccttctttgtgcccgtaggttgaaccggcctcttactctctcagagaagattgtatatggacacctggatgacccagccaaccaggagatcgagcggggaaagacatacctgcgtttacggcccgaccgggtggccatgcaggatgccacagcccagatggctatgctacagttcattagcagtgggctgcctaaggtggctgtaccatcaaccatccactgtgatcatctgattgaggcccaggttgggggtgagaaagacctgcgcagggccaaggtgagcccaagagggctggtggctgggtgtggagtgggggaagggagacagagcccaggctgcagaaggctgaggcactactgctgagctgcctgtcAGTCTTTCCATGGCACTGATGGTCTGAGGATGGAATCCAGACCTCATATGTGACAAGCAAGTGTTCTACCACTGAGCCATTCCCCAGCCCTGAGGGTTAACATCTTTTTAAGACTTGCTTTTAATTCTTCCTAATACTTAAAATGAAAATCTGATGCAAA

This mutation is a 418 bp deletion beginning at Chromosome 15 position 81,895,108 bp and ending after 81,895,525 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
53935 TGC AAA CAA GCA GAA CTT GG Common A
53936 GAA GTA GCT TTG GGA CTC AGC Wild type Reverse A
53937 CCA TCC TCA GAC CAT CAG TG Mutant Reverse A
53938 Fluorophore-1 AGA GGG AAA AGC TGT GCC AG Quencher-1 WT Probe
53939 Fluorophore-2 CTG CCA CAC AAG TCT TTC CAT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
53935 0.40 uM
53936 0.40 uM
53937 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.