Protocol 35850: Probe Assay - Hars2<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

>chr18:36788638+36788734 97bp CCGATCGAATTGGGGACT AGTTCAACAAACACACATCACC

Mut= 93 bp

Wt= 97 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case and insertion with carrots ^g^ (CTAC insertion):

 AAGGTAAGAAAGAAAACATGCTGTAAATAGATCTTATCTGGGTTTCTGCCCTGCCCTCAAATCCCTGTCATTAGGGAAAGAGTAAGGAAACCATGGCTAGAAGTGGCTATGCATGAGAAGGAAAAAGGCTAAGGAGACATTTTAGAACAAGGTCCGAGTTACACCATGCAGTGGGGACTGTGGCTGGATTTCCTGAGTTGCCTC^gggctttactgaattcactgttagtcccttaagacatccataccaaaggaaaatgagaggtcctgcttcagttagagaaagggcggagctaatgtttggatcttaatgcagatgtcttgggaaggcgtgaggcatgagatggtggcaaagaaaggtctagctcccgaggtggccgatcgaattggggacttcgtccaatatcatggtaagaaccagggtttttag^AGCCATGATAGAACAGGTTGAGGGTGATGTGTGTTTGTTGAACTTCTACCTCTAGATCAGTTCCAGAGTTTAGCCTGGATAAAGGTTCCCTTTTATGTATTCTAACTGCTTTATATCTTCTTAGTAAAGGCAGAAACAGAAGAAGGTCTAGCCATGAGACCAAGGCTGCAGTCCTTTTAAAGAAAATGAGGAAGACTGGCTGGAGCATAT

This mutation is a 225 bp deletion beginning at Chromosome 18 position 36,788,466 bp and ending after 36,788,690 bp (GRCm38/mm10). In addition, there is a 4 bp insertion at the deletion site (CTAC).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47158 CCG ATC GAA TTG GGG ACT Wild type Forward A
47159 AGT TCA ACA AAC ACA CAT CAC C Common A
47160 TAC ACC ATG CAG TGG GGA CT Mutant Forward A
47161 Fluorophore-1 CAA TAT CAT GGT AAG AAC CAG GGT Quencher-1 WT Probe
47162 Fluorophore-2 CCT GAG TTG CCT CCT ACA GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47158 0.40 uM
47159 0.40 uM
47160 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.