Protocol 31247: Probe Assay - Tyr<c-ch> 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=  97 bp

Wild Type = 97 bp

GGCTtcttgGGGCA(G/A)CACTGGTGGGA

Mut =A

WT= G

>chr7:87429155-87429251 97bp ACTTGGAACAAGCCAGTCGT AGCCTACTGCTAAGCCCAGA

 

Sequence

Wt Sequence:

 

Mutant Sequence:

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36462 ACT TGG AAC AAG CCA GTC GT Forward A
36463 AGC CTA CTG CTA AGC CCA GA Reverse A
36464 Fluorophore-1 CTT GGG GCA GCA CTG GT Quencher-1 WT Probe
36465 Fluorophore-2 TTC TTG GGG CAA CAC TGG T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36462 0.40 uM
36463 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

Stock Number Strain Name
034245 FVB(129P2)-Pde6b+ Gtf2iem2Jdd Gtf2ird1em2Jdd Tyrc-ch/Mmjax
034672 FVB(129P2)-Pde6b+ Gtf2ird1em3Jdd Tyrc-ch/Mmjax
004624 FVB.129P2-Pde6b+ Tyrc-ch Fmr1tm1Cgr/J
004828 FVB.129P2-Pde6b+ Tyrc-ch/AntJ
038293 FVB.Cg-Pde6b+ Tyrc-ch Ccdc198em1Iad/J
000271 SH1/LeJ
6 strains use this protocol