Protocol 32472: Probe Assay - Asb17<em1(IMPC)J>alt1
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

>chr3:153850467+153850562 96bp ACACAGCAGCGACACACAG AGGAAAGAAAACTGCCACCA

Mut= 113 bp

Wt= 93 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence: (deleted region in lower case)

 

AGTTCACACAGATATAGGCACACGTAACTAAAAAAAATCAAATCTTTAAAAAATAAGCAATAAGAAAACTGTTTTTATTTTTAATTTTCATGTTATATTTGCAGAGCATGGTAAGGTTTATGCCCCCACCCGTGGGGGAGACACAGCAGCGACACACAGCTGCAGCGACTAAGTGCTTTGCCCATGTGGTGTGATGTgaaaggcggcttcagcatttggtggcagttttctttcctccttcagtttggtccacagttacaatgtcaaatccttcttaagacattacaggtgaacgttagttacatggtggtcactctgcttttggcttgcagaaccttcacacctgtgtactgccccagccccctgagtggcatcacacctctactctacgtggctcagacaagacagtcaaatatcttaaaaattctcctgcagtatggaatcctagaaagagaaaaaaaccctatcaacattgttctgacaatactactttacccttcgagagtgagaataatggttgaccacgagttgattgacattcaagaagatgccaagacatgtttaatgctatgttccagagtgctttctacgatctcagtcagggagatagaggtaagcggagcaccgcccttctcagggcgtggggagtggataataagagaagcagaggaacacttttggggtttaagcagagctatattctatggggaaaagccgagtgttgctctttggggtgcaatttaccatcagtaagtgttaggacataattgcaaggGAAAATTGCAGAGTATTTTTAATTTATGCATTATTTCCAATGGTGAATTAATGACCTCGCATATGCTCAGTAATTTAAAATGAGGCAAAATTCAAATTAACTAAAATTAGTCCTTCAAATGTTGATTTTTTTTGTTACCGTGAACTTCAAAATGGCAAAGCTGAGTGTCCTATAGACA

This mutation is a 578 bp deletion beginning at Chromosome 3 positive strand position 153,850,524 bp and ending after 153,851,101 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38437 ACA CAG CAG CGA CAC ACA G Common A
38438 AGG AAA GAA AAC TGC CAC CA Wild type Reverse A
38439 GGT CAT TAA TTC ACC ATT GGA Mutant Reverse A
38440 Fluorophore-1 AAA GGC GGC TTC AGC ATT Quencher-1 WT Probe
39176 Fluorophore-2 TGT GAT GTG AAA ATT GCA GAG TAT T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38437 0.40 uM
38438 0.40 uM
38439 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.