Protocol 35031: Probe Assay - Ccdc172<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

>chr19:58527763+58527865 103bp TGAGCAGTGTCAGATTCTTCAG CACCATGCTGTCTTCTGGTC

Mut= 98 bp

Wt= 103 bp

Fam=Mut

Hex=Wt

Sequence

Mut Sequence:

 tggtcacagcgattgattctttctaagttgtgagactagaagcctgctccttgaaacaagctgtttgatgtcttatgagaccagaagacagcatggtg

This mutation is a 2258 bp deletion beginning at Chromosome 19 position 58,525,572 bp and ending after 58,527,829 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45078 TGA GCA GTG TCA GAT TCT TCA G Wild type Forward A
45079 CAC CAT GCT GTC TTC TGG TC Common A
45080 TGG TCA CAG CGA TTG ATT CT Mutant Forward A
45081 Fluorophore-1 ACA CTG TGA ACT GAG CAT TTG ATT Quencher-1 WT Probe
45082 Fluorophore-2 AAG CCT GCT CCT TGA AAC AA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45078 0.40 uM
45079 0.40 uM
45080 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.