Protocol 44026: Probe Assay - Stmn2<em5/6(STMN2)Lutzy>-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 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

>chr3:8515051+8515188 138bp TGGATTACAGAATATTTCACTTCCAA AGGCCTTCTGTTGAGTCCTG

Mut= 89 bp

Wt= 138 bp

Fam=Mut

Hex=Wt

The WT Probe  (primer 64602) anneals over the nucleotide sequence containing mouse genomic variations rs37789913.

Sequence

Wt Sequence:

atgagcatgtgcccctcagaggaagtccactgtcctgatgagcatgtgcccctcagaggaagttccactgatgatgagcatgtgcccctcagaggaagttccactgtcctgacttttaaatctgtggcaatggattacagaatatttcacttccaaactcatatgcatagggatttgattcctctgaggatatgtattctgtgccatcactctctcttaattagattgtttaaaatatatttatattacaggactcaacagaaggcctttgaggaaaatggtgggagaaataagcctggctctgtgaatatgtgtgtgggtgggtgggtctgtgactcagttaagaaaaataggggactttcaggatagcatttgaaatgtaaatgaagaaaatatccagaaaaaattgttaaataaaaaagaagggataatatataatatgatattactgtcacttacaaatgatacacagaacagaagttaaaaccagaacctt

Mutant Sequence:

GGAAGTCCACTGTCCTGATGAGCATGTGCCCCTCAGAGGAAGTTCCACTGATGATGAGCATGTGCCCCTCAGAGGAAGTTCCACTGTCCTGACTTTTAAATCTGTGGCAATGGATTACAGAATATTTCACTTCCAAACTCATATGCATAGGGATTTATTACTCTGGGAATTATGTGTTCTGCCCCATCACTCTCTCTTAATTGGATTTTTAAAATTATATTCATATTGCAGGACTCGGCAGAAGACCTTCGAGAGAAAGGTAGAAAATAAGAATTTGGCTCTCTGTGTGAGCATGTGTGCGTGTGTGCGAGAGAGAGAGACAGACAGCCTGCCTAAGAAGAAATGAATGTGAATGCGGCTTGTGGCACAGTTGACAAGGATGATAAATCAATAATGCAAGCTTACTATCATTTATGAATAGCAATACTGAAGAAATTAAAACAAAAGATTGCTGTCTCAATATATCTTATATTTATTATTTACCAAATTATTCTAAGAGTATTTCTTCCTGAATACCATGTGAGAAAATTCTTAAGAATTTATTGAGTATATTAAGCATTCACTGGGTTATAAGGAATTCACTAGGTATGACCCTATTT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50411 TGG ATT ACA GAA TAT TTC ACT TCC AA Wild type Forward A
64599 AGG CCT TCT GTT GAG TCC TG Wild type Reverse A
64600 TTG GCT CTC TGT GTG AGC AT Mutant Forward A
64601 ACA AGC CGC ATT CAC ATT C Mutant Reverse A
64602 Fluorophore-1 CTG TGC CAT CAC TCT CTC TTA ATT AGA Quencher-1 WT Probe
64603 Fluorophore-2 AGA CAG ACA GCC TGC CTA AGA AGA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50411 0.40 uM
64599 0.40 uM
64600 0.40 uM
64601 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.