Stock No: 002313
Protocol 21045: Probe Assay - Emv30<b> 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

Mutant=  100 bp

Wild Type = 130 bp

Sequence

Wt Sequence:gatctctgtgagttcaaggccagcgtggtctacagagggaaagtagcaagtttcaggatatCCAGggctacagaatagagagactctgtcttcaagaaaatgaattaaattaattaaaatgtttaaagttatgtgttttca

ccttcctaattttctttttaaaaatttattttatccagctgggcaatgtagcacatgcc

 

Mutant Sequence: GCCAGCGTGGTCTACAGAGGGAAAGTAGCAAGTTTCAGgatatccagtgaaagaccccTTCATAAGGCTTAGCCAGCTAACTGCAGTAACGCCATTTTGCA

AGGCATGGGAAAATACCAGAGCTGATGTTCTCAGAAAAACAAGAACAAGGAAGTACAGAGAGGCTGGAAAGTACCGG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
31314 AGA AAA TTA GGA AGG TGA AAA CAC A Wild type Reverse A
31588 GGT CTA CAG AGG GAA AGT AGC A Common A
31589 CAT GCC TTG CAA AAT GG Mutant Reverse A
31590 Fluorophore-1 ATA TCC AGG GCT ACA GAA TAG AGA GA Quencher-1 WT Probe
31591 Fluorophore-2 TCC AGT GAA AGA CCC CTT CAT A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
31314 0.50 uM
31588 0.50 uM
31589 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
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.