Protocol 43336: Probe Assay - Siral2<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

  
>chr15:85050415+85050512 98bp GGAAACAGAACCAGCTACAACC TGTCACAGCAGCTAATTGTGG

Mutant=  94 bp

Wild Type = 98 bp

Sequence

Large deletion:  Mutant sequence with junction in uppercase

ggaaacagaaccagctacaaccccacaacaatatagaattaacTGcagcgttcctgtcagtgatagttaaaccttggaaggcttatgtggctta

This mutation is a 3638 bp deletion beginning at Chromosome 15 position 85,050,459 bp and ending after 85,054,096 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
62819 GGA AAC AGA ACC AGC TAC AAC C Common A
62820 TGT CAC AGC AGC TAA TTG TGG Wild type Reverse A
62821 TAA GCC ACA TAA GCC TTC CAA Mutant Reverse A
62822 Fluorophore-1 CTG CAT ATA ACT AAG AAG GTC CCT CC Quencher-1 WT Probe
62823 Fluorophore-2 AGA ATT AAC TGC AGC GTT CCT GTC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
62819 0.40 uM
62820 0.40 uM
62821 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.