Protocol 45296: Probe Assay - Arid3c<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

>chr4:41728303-41728383 81bp TTCTCCTCTCCCTGTATTTGC TCTATCTACGCGACCTGTCC

Mutant=  100 bp

Wild Type = 81 bp

Sequence

Wt Sequence ttctcctctccctgtatttgccacgtgcaaggtctgagctaCctagaggcaccactagctaggacaggtcgcgtagataga

Mut Sequence:

ttctcctctccctgtatttgccacgtgcaaggtctgagctaCACTCATTAACCTGGtgagatcaattaggaaggtgggagggagaactggtcaggactgg

A 517 bp deletion beginning at Chromosome 4 position 41,727,825 bp and ending after 41,728,341 bp (GRCm38/mm10). This mutation deletes ENSMUSE00000527822 (exon 3) and 444 bp of flanking intronic sequence including the splice acceptor and donor and is predicted to cause a change of amino acid sequence after residue 103 and early truncation 3 amino acids later. There is a 13 bp insertion (ACTCATTAACCTG) at the deletion site. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65211 TTC TCC TCT CCC TGT ATT TGC Common A
65212 TCT ATC TAC GCG ACC TGT CC Wild type Reverse A
65213 CCA GTC CTG ACC AGT TCT CC Mutant Reverse A
65214 Fluorophore-1 AGC TAC CTA GAG GCA CCA CTA GC Quencher-1 WT Probe
65216 Fluorophore-2 CAT TAA CCT GGT GAG ATC AAT TAG GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65211 0.40 uM
65212 0.40 uM
65213 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.