Protocol 35745: Probe Assay - Abca8a<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

>chr11:110072010-110072095 86bp AAGGAAGCAGCCTTTCCTCT GCTTCACTTAGCTGTGGTTAGACA

Mut= 80 bp

Wt= 86 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TTAGTATTACTCCTTTCAGAGTTCTAAGAAATAGCTATATTTGTTGGCTAGTTAATGTTTAGACTCCAATGACGGCTAGCACCTCTGTTCATGGCATTTCTTCTTGTTGCCCTTCGATACTGCCTGGCTGACCTGAAGAGATTCTGGACTTTATGCAGatgttttttctaacatctgtaaagtgttagtttcctactttgccttgctatggtagctgtccacattgtatcattttctagcttaccctatcactacgtatggttcagctcaggaaaaatgctcacagtggcttgaacagtgtccaggtctcagtccacaccaaaggaagcagcctttcctctaggtaagatcaggctttaaaacactggtccaagtaccattgctgtctaaccacagctaagtgaagctcacatgccatcccttatatccttgcaagccaaatctgcgtaaagatttcagccagactaggagttagatagtaaagaaaaccattgctccttaaacagtttatctcttgctctctttctcagaatcagaaatcttacaaaagactacatacaaaagtctaagcgcacagaagctttgaaaggtaagtaactaaggtagaaaaatatggtttttagtcagttaacttttctctccatgagcaattctagactgtgtaagtggatgttgagtctcctgaccccccaaaccccacaactgagctaacattgacaggctgtgtgattttgagtattaactttgagtattaattttgatttaataagtgaatgaatgtatacttttataaattctagtcacactataaatacgtaggcactatatgagccctgaattaaacctacatttgtgattttataccttcatgctgttttcctcccagatctgacactggatgtatataaaggccagattactgcgatccttggtcacagtggagctgggaaatcaacactgttaaatgtcctgagtgggttgtgtgttcccaccaaaggtaagagacagagtctttcctctaagttggcaatcatctgtgcttttgttagacatttgagcattaaggtcagacagagataaactcccagctattgacacataatctctattactcttttataaaatgtatatgatattaactgaaatgtacagaattttagtattttagaatactcaaaattttatagaattaacagtttcaattgatattgatcacatttttctgtatagataaagtaaccttgaaaattagcaacttttaaaatatgACTGGAAAGGTCTCTCAATCTCACTGTTTTGACTGTAACTTATAACCTAGAATGGCTTTACCTACATGTGTGAGACTTTAGCTGGGCTATTCTCCCCTTCCCCTTCTCCCCTCCCTTCCCTCTCTATCTCCTTCACCCTCTCTATCTCCTTCATCCCCTCCCTCTCCTTCTCCCTCTTCCCTTTCTCC

This mutation is a 1110 bp deletion beginning at Chromosome 11 position 110,071,149 bp and ending after 110,072,258 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46922 AAG GAA GCA GCC TTT CCT CT Wild type Forward A
46923 GCT TCA CTT AGC TGT GGT TAG ACA Wild type Reverse A
46924 GAT ACT GCC TGG CTG ACC TG Mutant Forward A
46925 TAC AGT CAA AAC AGT GAG ATT GAG AG Mutant Reverse A
46926 Fluorophore-1 TCA GGC TTT AAA ACA CTG GTC C Quencher-1 WT Probe
46927 Fluorophore-2 CTG GAC TTT ATG CAG ACT GGA AA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46922 0.40 uM
46923 0.40 uM
46924 0.40 uM
46925 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.