Protocol 44280: Probe Assay - Cyp2w1<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=  128 bp

Wild Type = 127 bp

  
>chr5:139353973+139354099 127bp CCATCTTCCAGCACATCCA TCCTGTCTTGCAATAGGCTGA

Sequence

Large deletion:  Mutant sequence with junction in uppercase

ccatcttccagcacatccagcgaggtgggggtaggtgggcattggggttccttgccagatgggggggagactaggCCtggagtgggcactgatgttgggtgaacccccagtaaacaccagtgctttcc

 

 

This mutation is a 1837 bp deletion beginning at Chromosome 5 position 139,354,049 bp and ending after 139,355,885 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65184 CCA TCT TCC AGC ACA TCC A Common A
65185 TCC TGT CTT GCA ATA GGC TGA Wild type Reverse A
65186 GGA AAG CAC TGG TGT TTA CTG Mutant Reverse A
65187 Fluorophore-1 TGG ATT GGT ACT AGG GAG AGA CAG Quencher-1 WT Probe
65188 Fluorophore-2 AGA CTA GGC CTG GAG TGG GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65184 0.40 uM
65185 0.40 uM
65186 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.