Protocol 45760: Probe Assay - Atosb<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

Mutant=  116 bp

Wild Type = 100 bp

>chr4:43037233-43037332 100bp CTGCAGAGCTTGTCCCAAA AGAATCCGTGCAGCCAAT

Sequence

Large deletion:  Mutant sequence with junction in uppercase

tctgttgagtcctcaggtagcccaattcttctgcatacgccctaggtctccagcctgcctcttctgtttctgcagagcttgtcccaaaGAtcttaggctgggatgagctgagtcttggaccatagtcttagtcctttttgtataaggtgctgggggctgggtgggtaaagtgaagctacagaggctaaactctgagaatctggattcttacactgattctgtagtttagccccat

 

 

This mutation results in a 4988 bp deletion beginning at Chromosome 4 position 43,032,325 bp and ending after 43,037,312 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66394 CTG CAG AGC TTG TCC CAA A Common A
66395 AGA ATC CGT GCA GCC AAT Wild type Reverse A
66396 Fluorophore-1 AGA TGT CTT GGT GTG TGT GCT G Quencher-1 WT Probe
66397 Fluorophore-2 AGG CTG GGA TGA GCT GAG TCT TGG Quencher-2 MUT Probe
66398 GCC TCT GTA GCT TCA CTT TAC CC Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66394 0.40 uM
66395 0.40 uM
66398 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.