Protocol 36291: Probe Assay - Stmn2<em2(delEx3)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

Mutant=  87 bp

Wild Type = 95 bp

Mutation description:  1028 bp deletion

>chr3:8545128+8545222 95bp CTCTCAGCCTCCAGGTTTCT GAGGAAAGGGTGCATTTGG

 

Sequence

Wt Sequence: agctgggcaaagcacagaaaggaggccgggaagcaacgtgcctccatgggctctgcttcactctcagcctccaggtttctgctgaagtctgagccctggcttccctcagtggTGGATTGTGTCGTGGAAGTGTAAGCCAAATGCACCCTTTCCTCCCAAGTTGGTTTTTGTCATGGTGTTTTGTCACAGTGA

Mutant Sequence: cttcactctcagcctccaggtttctgctgaagtctgagccctggcttccctcagtgGTggagggcagaggttggaaagcccagtaaagagcacttcccaagttccattccaagcatccatttcaag

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48340 CTC TCA GCC TCC AGG TTT CT Common A
48341 GAG GAA AGG GTG CAT TTG G Wild type Reverse A
48342 Fluorophore-1 TGG ATT GTG TCG TGG AAG TGT A Quencher-1 WT Probe
48343 TGC TCT TTA CTG GGC TTT CC Mutant Reverse A
48344 Fluorophore-2 CCT CAG TGG TGG AGG GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48340 0.40 uM
48341 0.40 uM
48343 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
036033 C57BL/6J-Stmn2em2Lutzy Tg(STMN2)9439Lutzy/Mmjax
033740 C57BL/6J-Stmn2em2Lutzy/Mmjax
2 strains use this protocol