Stock No: 031558
Protocol 32208: Probe Assay - Sumf1<em8(S153P,F154F,F156F)Lutzy>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

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

Mut= 117 bp

Wt= 117 bp

Sequence

Wt Sequence:TGCCCTGACTCTAACATTTTCTGGCTTTTTCTTTTTTACTCTCCTTATAGGCTGAGAAGTTTGGAGACtCTTTcGTCTTtGAAGGCATGTTGAGCGAGCAAGTGAAAACGCATATCC

 

Mutant Sequence:TGCCCTGACTCTAACATTTTCTGGCTTTTTCTTTTTTACTCTCCTTATAGGCTGAGAAGTTTGGAGACcCTTTtGTCTTcGAAGGCATGTTGAGCGAGCAAGTGAAAACGCATATCC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38602 TGC CCT GAC TCT AAC ATT TTC TG Forward A
38603 GGA TAT GCG TTT TCA CTT GCT Reverse A
38607 Fluorophore-1 TGG AGA CTC TTT CGT CTT TGA A Quencher-1 WT Probe
38608 Fluorophore-2 TGG AGA CCC TTT TGT CTT CG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38602 0.40 uM
38603 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 4.0 -- 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.