Stock No: 000486
Protocol 30745: Probe Assay - Fas<lpr> 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

>chr19:34312593+34312684 92bp GTGCCCTAGGAAACACAGCA CTCTAGCCAGGAATACAAATGACA

Mutant=  110 bp

Wild Type = 92 bp

Sequence

Wt Sequence: gtgccctaggaaacacagcatagattccatttgctgctgtgtccatgtGGagcagatttgactgcgtttgtcatttgtattcctggctagag

 

Mutant Sequence: gtgccctaggaaacacagcatagattccatttgctgctgtgtccatgtgTGTAGTCTCCCCTCCCCCAGCCTGAAACCTGCTTGCTCGGGGTGGAGCTTCCTGCTCATTC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35342 Fluorophore-1 TGT GTG TAG TCT CCC CTC CC Quencher-1 MUT Probe
35343 Fluorophore-2 ATG TGG AGC AGA TTT GAC TGC Quencher-2 WT Probe
35344 GTG CCC TAG GAA ACA CAG CA Common A
35345 GAA TGA GCA GGA AGC TCC AC Mutant Reverse A
35346 CTC TAG CCA GGA ATA CAA ATG ACA Wild type Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35344 0.40 uM
35345 0.40 uM
35346 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.