Protocol 30079: Probe Assay - Aacs<em1J> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

>chr5:125963064+125963186 123bp AGCTCCTCCCAGACATTCAG GCAAAACAGCAACTCTTCAGC

Mutant=  108 bp

Wild Type= 123 bp

Sequence

Wt Sequence: agctcctcccagacattcagggccatcagagtcaccgagtccatggcacgtgacatccttaggcctgtggctttccaccaaggggccaccctttgaggctcggctgaagagttgctgttttgc

 

Mutant Sequence: agctcctcccagacattcagggccatcagagtcaccgAGttgcttgaggaataccgaatcatacctgctagtcccatgtgtgggccagcaacatggctcagtgggtaa

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
33848 AGC TCC TCC CAG ACA TTC AG Common A
33849 TTA CCC ACT GAG CCA TGT TG Mutant Reverse A
33850 GCA AAA CAG CAA CTC TTC AGC Wild type Reverse A
33851 Fluorophore-1 CCT TAG GCC TGT GGC TTT C Quencher-1 WT Probe
33852 Fluorophore-2 TGC TTG AGG AAT ACC GAA TCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
33848 0.40 uM
33849 0.40 uM
33850 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.