Protocol 34114: Probe Assay - Tubb2b<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

chr13:34128761-34128880 120bp TCGTGTTCGGTGAGTATGTG CTTAGGCGCCACTGCTATGT

Mut= 118 bp

Wt= 120 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGCAGCTGTTCTCATGGGAAACCGCCCAGTTTCAACTTTCTGATCCTCTACAATGCAAGCCCTAAGGCTGTGCCTGGGTCATGCCACTCATTAAAGCTTCGGCAACGTCCTGTTGTCAAAACTGTGGGTGATGGGGGGGCTGCTCATTGAGAttgggggtagggttgtgcatagctaaatacagtctttttatgtcacagttttgggaggtcatcagtgatgagcatggtatagaccccactggaagttaccatggagacagtgatttgcaactggaaagaatcaatgtatactacaatgaagcaactggtaattacttttaacaccccatcttcaattctggcatccctaagctctgctcttatttgaacccaggagaaggtggcttattttaggatgaccatttttggggtggggtagcctcttctgtgccaaccaaaccctttgaaagcttggtcataatagacctttctttgtttgggggtaccaccaaacaggaaggaggtgggaccgagccctactctatagaagatactgcctggagggctaagccagctcttgttcttctgcaggtaataaatatgtgcctagggccatcctggtggacctggagccaggcacaatggactcagtcaggtctggaccatttgggcagatcttcaggccggacaacttcgtgttcggtgagtatgtggcatagctgaagacagaagagcacaaggtgttaattatcactagcacaaagggTGGAGGAGTGTGATAGACAGGGAGCATACATAGCAGTGGCGCCTAAGCCTTATCAGGCTTTGAGGCAATTGCATCTTAGCTTTTTCAAAGGCAGGTGTGCTCTGTTAACTCATTTATAATTGTATTGATAACCAAATAGTTTATTGCTTTATT

This mutation is a 595 bp deletion beginning at Chromosome 13 position 34,128,808 bp and ending after 34,129,402 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42858 TCG TGT TCG GTG AGT ATG TG Wild type Forward A
42859 CTT AGG CGC CAC TGC TAT GT Common A
42860 TGC CAC TCA TTA AAG CTT CG Mutant Forward A
42861 Fluorophore-1 CTG AAG ACA GAA GAG CAC AAG GT Quencher-1 WT Probe
42862 Fluorophore-2 CTG CTC ATT GAG ATG GAG GAG T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42858 0.40 uM
42859 0.40 uM
42860 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.