Protocol 34948: Probe Assay - Abca2<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

>chr2:25435759+25435858 100bp AGAGATAAGCTTGACTTCACGAG ACAGAAAAGCTGCCAAGTGC

Mut= 95 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Mut Sequence:

 taaccagttggcctgagaggtcagaggtcactgctaaggggggtgcctcctgtgctttagggttagaggtgcagtggcacttggcagcttttctgt

This mutation is a 2972 bp deletion beginning at Chromosome 2 position 25,432,845 bp and ending after 25,435,816 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44878 AGA GAT AAG CTT GAC TTC ACG AG Wild type Forward A
44879 ACA GAA AAG CTG CCA AGT GC Common A
44880 TAA CCA GTT GGC CTG AGA GG Mutant Forward A
44881 Fluorophore-1 TCT AGG TAC TGG TGT GCC TGC Quencher-1 WT Probe
44882 Fluorophore-2 AGA GGT CAC TGC TAA GGG GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44878 0.40 uM
44879 0.40 uM
44880 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.