Protocol 36267: Probe Assay - Tuba1c<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

>chr15:99033984+99034082 99bp TTGAAGTATAGTCCTAGGCAGGAG GGGAAGAACGTGTGAGAATG

Mut= 98 bp

Wt= 99 bp

Fam=Mut

Hex=Wt

Sequence

Mut Sequence:

ttgaagtatagtcctaggcaggagtggccatgatatgacagccatcactgtccctaactgaatcatggattcatgactcattgagtgtgcccagttga

This mutation is a 4195 bp deletion beginning at Chromosome 15 position 99,034,012 bp and ending after 99,038,208 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48219 TTG AAG TAT AGT CCT AGG CAG GAG Common A
48220 GGG AAG AAC GTG TGA GAA TG Wild type Reverse A
48221 TCA ACT GGG CAC ACT CAA TG Mutant Reverse A
48222 Fluorophore-1 CCA TGC CCC AAG GTC TC Quencher-1 WT Probe
48223 Fluorophore-2 ACA GCC ATC ACT GTC CCT AAC T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48219 0.40 uM
48220 0.40 uM
48221 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.