Protocol 30524: Probe Assay - Rab8a<em1(IMPC)J> 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

>chr8:74692133+74692229 97bp GACAGGAGCAGCAGAAAGGT TTCCCTGCTAGAAGCACAGC

Mutant=  111 bp

Wild Type = 97 bp

Sequence

Wt Sequence: gacaggagcagcagaaaggtggagcgcatctcccattcccagcttcCCttcagctactgttgtcgccacagccttaggctgtgcttctagcagggaa

 

Mutant Sequence: gacaggagcagcagaaaggtggagcgcatctcccattcccagcttcCCtgaatcgtggcccttcgctggactctacactgggagcagagctgttggtgcccttccacatat

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34815 GAC AGG AGC AGC AGA AAG GT Common A
34816 TTC CCT GCT AGA AGC ACA GC Wild type Reverse A
34817 ATA TGT GGA AGG GCA CCA AC Mutant Reverse A
34818 Fluorophore-1 AGC TAC TGT TGT CGC CAC AG Quencher-1 WT Probe
34819 Fluorophore-2 CCC TTC GCT GGA CTC TAC AC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34815 0.40 uM
34816 0.40 uM
34817 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.