Stock No: 004155
Protocol 32425: Probe Assay - Alox5<tm1Fun> Probe Alternate1
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

Mutant=  88 bp

Wild Type = 102 bp

Sequence

Wt Sequence: tgagaagatcccgatacctacaaagatttgtcctcctgcaggatcgtgggatcctgcccagcggtcctatctagaggtcattctctccacagAGCGAGTCAAGAACCACTGGCAGGAAGACCTCATGTTTGGCTACCAGTTCCTGAATGGCTGCAACCCAGTACTCATCAAGCGtTGCACAGCGTTGCCCCCGAAGCTCCCAGTGACCACAGAGATG

 

Mutant Sequence: gcaaaaccaaattaagggccagctcattcctcccactcatgatctatagatctatagatctctcgtgggatcattgtttttctcttgattcccactttgtggttctaagtactgtggtttccaaatgtgtcagtttcatagcctgaagaacgagatcagcagcctctgttccacatacacttcattctcagtattgttttgccaagttctaattccatcagaagcTACTCATCAAGCGTTGCACAGCGTTGCCCCCGAAGCTCCCAGTGA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
23994 CAG CCT CTG TTC CAC ATA CAC T Mutant Forward A
39085 Fluorophore-1 GCT GTG CAA CGC TTG ATG Quencher-1 Common A
39086 TCT CTC CAC AGA GCG AGT CA Wild type Forward A
39087 Fluorophore-2 TTG CCA AGT TCT AAT TCC ATC AGA AG Quencher-2 MUT Probe
39088 Fluorophore-3 AGA CCT CAT GTT TGG CTA CCA GTT C Quencher-3 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
23994 0.40 uM
39085 0.40 uM
39086 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.