Protocol 32964: Probe Assay - Cdc23<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

>chr18:34644899-34645023 125bp AGCCTGTGCTGACGGAGAGT GTTGCAGAAAGCAAGCATGA

Mut= 117 bp

Wt= 125 bp

Fam=Mut

Hex=Wt

Sequence

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

 GTGTTTCACCACGCCCGGCTTTCATATTTTTACATATCTGTAGTTCATTACTATATTTAAGTGTTTGGTGCTTAATTTATTCCATATTTATTCAGTGGGAACACCTGAATGAATGGTTGTCTATAAGGCGGATGTTGTT^ttgcttaggcattagttagccctgtagatagaaccagctgctggttttattaataagccagacaacaacaaaaatcatcaacaaaaacccaaaccctattcgttgatttctgtcttcccccttacagtctggagagaagaagaaggatgacgaaacagttgatagcctaggtatgaccccccaccccccatgcaggaaccctaagaccgcacagcctgtgctgacggagagtggcttccctcagccactgctgtcatccatctctacccg^AGGGCTGCTGTGGGAATCAGCCTGTGGGTATGCTTGCCCCGCTTGAATCATGCTTGCTTTCTGCAACCTAAGGTGGAAAATTGTTTTAACTGAAACTTCTGAGTTGTAAACATTGATAAGTCGGACTACACCAGCCAT

This mutation is a 270 bp deletion beginning at Chromosome 18 position 34,644,966 bp and ending after 34,645,235 bp (GRCm38/mm10). In addition, there is a 7 bp insertion (GTCTATA) at the deletion site that will not alter the results of the exon deletion

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40359 AGC CTG TGC TGA CGG AGA GT Wild type Forward A
40360 GTT GCA GAA AGC AAG CAT GA Common A
40361 GGG AAC ACC TGA ATG AAT GG Mutant Forward A
40362 Fluorophore-1 TGC TGT CAT CCA TCT CTA CCC Quencher-1 WT Probe
40363 Fluorophore-2 TGT TGT TGT CTA TAA GGG CTG CT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40359 0.40 uM
40360 0.40 uM
40361 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.