Stock No: 031423
Protocol 32200: Probe Assay - Sumf1<em3/4(A277V)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= 112 bp

Wt= 112 bp

Sequence

Wt Sequence:AGCATTATGCCAACATCTGGCAGGGCAAGTTTCCTGTGAGCAACACTGGCGAGGATGGCTTCCAAGGAACTGccCCCGTGAGTAAGCAGGCTTCTTGAGAAGGGCTGTGTGC

 

Mutant Sequence:AGCATTATGCCAACATCTGGCAGGGCAAGTTTCCTGTGAGCAACACTGGCGAGGATGGCTTCCAAGGAACTGtaCCCGTGAGTAAGCAGGCTTCTTGAGAAGGGCTGTGTGC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38585 AGC ATT ATG CCA ACA TCT GG Forward A
38586 GCA CAC AGC CCT TCT CAA G Reverse A
38587 Fluorophore-1 AAC TGC CCC CGT GAG TAA G Quencher-1 WT Probe
38588 Fluorophore-2 CCA AGG AAC TGT ACC CGT GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38585 0.40 uM
38586 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.