Protocol 35782: Probe Assay - Stmn3<em1(IMPC)J>
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

>chr2:181308518-181308634 117bp CTGAGCAATTGGAATGAGATG ATTCAACCTGCCCTCCCTCT

Mut= 120 bp

Wt= 117 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGATGGAAGCAGACAGATAAAGAATTCAAAGTCATTGTAGGCTACAAAGTGAATTTAAGGCCAGTCGGATTACAGGAGACCCCTTCACAACACAAAACAAATATGCACATAGATAAAGGCCACTGTAGAATACAAAGACCTAGAAATACcacaggatcatggtacctgctcaccttgactttacatccacagcctacaaggagaagatgaaggagttatctgtgctgtcgctcatttgctcctgcttctactctcagcctcacccgaacaccatctaccagtatggaggtgagtgtatccacacagctgctggccctgctcagctagggcctgacaaccttacttctctcgtttcttctggcctctggcaatgctgtgaactggctggagagagtcattctgggaggaggggcctatgctaaagagtaacggcaccccttcttaacccttgttgggatgtgtgctggaaataaacctctcatcctgcctgtatgcccaaaccaggcctcctgaagtcccatctggtcttgtcctacagatatggaagtgaagcagctggataagcgggcctctggccagagcttcgaggtcatcctcaagtctccttctgacctatctccagagagccctgtgctctcttctcctcccaagaggaaggatgcttccttggaagagctgcagaagcggctggaggcagctgaggagcggaggaaggcaaggactctccttacacacttgacattgctgaccaggccctgtgcttggtcagggcattgggctgggtagtgcagaacttactgctgagcaattggaatgagatgaaagatggatgtgccaagatggtaacaaacacagggcaataggaacCACATTCTTGAGGGGGACCTCGATCATGGCAGAGGGAGGGCAGGTTGAATTCCCCCAGAAAGAGCCCTTAAGACATAAAGCTGAGGCTAGTGCTCCTTTCAGGACAGAAGATCGAGGAGAAAAGGTCTCACATCTAAAGACAGTTAGCTGGGTTGGGTA

This mutation is a 718 bp deletion beginning at Chromosome 2 position 181,308,568 bp and ending after 181,309,285 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47011 CTG AGC AAT TGG AAT GAG ATG Wild type Forward A
47012 ATT CAA CCT GCC CTC CCT CT Common A
47013 CCC CTT CAC AAC ACA AAA CA Mutant Forward A
47014 Fluorophore-1 ATG GAT GTG CCA AGA TGG TAA Quencher-1 WT Probe
47015 Fluorophore-2 AGG CCA CTG TAG AAT ACA AAG ACC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47011 0.40 uM
47012 0.40 uM
47013 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.