Protocol 45732: Probe Assay - Armc10<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

Mutant=  110 bp

Wild Type = 120 bp

>chr5:21648737+21648856 120bp TGGGAAATAATGCAGCCTTC GCTGTGAGCACAAGTGGCTA

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case; insertions with carrots ^g^ (g insertion):

AAAGTGTAAGCAGGCGTTCCCACACACTGCACCTCCCTGGTCCTCAGTAGC^ccccccc^gcctcccctcccacgggccagcctccatggctgacaccacttgcatttttaaagccatgaccttccttcttcctcttctcccatctccctcttcctagcagatgccaagatttgtagcttgcctgtcctgtggtttttctcttaacacatgaaaattgtccatgacctttaaaaagccaaacaactgtgcccagtatcagagtatatgtttgaacatttcacacattaacaataaaacacattactggtgagtttcaatgaattctaaatggcttccttcttatgcagaagacctaaccgatggctcctatgacgatatcttaaatgcagagcagcttaagaaacttctgtatctgctggagtcaaccgacgatcctgtcattactgaaaaggccttggtcaccttgggaaataatgcagccttctccactaaccaggtaagcctcctctttccacacaagtagctgtggccccattctgaaCTCTTAGCTATCCTCATATGGCATAGCCACTTGTGCTCACAGCAGTGTTTG

This mutation is a 482 bp deletion beginning at Chromosome 5 position 21,648,332 bp and ending after 21,648,813 bp (GRCm38/mm10).

There is a 7 bp (CCCCCCC) insertion at the deletion site.  

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66324 TGG GAA ATA ATG CAG CCT TC Wild type Forward A
66325 GCC AAA AGA AAA GTG TAA GCA G Mutant Forward A
66330 GCT GTG AGC ACA AGT GGC TA Common A
66331 Fluorophore-1 TCC ACA CAA GTA GCT GTG GC Quencher-1 WT Probe
66332 Fluorophore-2 CCC ACA CAC TGC ACC TCC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66324 0.40 uM
66325 0.40 uM
66330 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.