Protocol 35045: Probe Assay - Arpc1a<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

>chr5:145095934+145096041 108bp AGAAGGAGGGGAAGAAACCA AGCACAGGTCACTTGCTACG

Mutant=  93 bp

Wild Type = 108 bp

Sequence

wt sequence, deletion in lower case

CCTGGGCTTCTTAACACCGTGTGCCAAAAGAAAACACAAATTCCCAGTTCTGTTCTACCTGGCTTTGTAAGGATCCTGCGTCCTCTGGTGTCCTTAGGAGAGAAACAGCCTTGGTTTTGTATGAGATAATAACCATAGATAGGAGAAGGAGGGGAAGAAACCACTAAGCCCAcagttaccccatgcccagctgtcacccgccaatagcccagtcccttgccactggccaggcgtagcaagtgacctgtgcttcacttttaggtattgactgggctcctaagagcgaccgtattgtcacttgtggggcagaccgcaacgcctatgtctggagtcagaaagatggcatctggaagcccaccctggtgatcctgaggattaaccgtgcagccacttttgtgaagtggtccccacttgagaacaagtttgctgtggggagcggagcccggctcatctctgtctgttactttgagtctgagaatgactggtgagccaagttgcatgtgtcctctcactctgctttgtatactccatgctgggatgctgtgctcctgggacctgttgaggctctgtgtactgcagtgcccttcctggcctggagaggctttctccctctcaaCTCGTATGTGCATGACTCAGCTCCCATACCTTTTCTTTGACTCTTCACTTCCCTTCCTTACTCCTCTACTTCCTTTTGTCCATTACTTCCTCCCTACCCCTTCTTTCCCTTCCCTCCTTCAGCTCCTTCATTTCCTCCTTCCAGCTCCCGACTTATCTTAATTCACATGGGCAACA

 

A 444 bp deletion beginning at Chromosome 5 position 145,095,963 bp and ending after 145,096,406 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45117 AGA AGG AGG GGA AGA AAC CA Common A
45118 AGC ACA GGT CAC TTG CTA CG Wild type Reverse A
45119 GGA GTA AGG AAG GGA AGT GAA GA Mutant Reverse A
45120 Fluorophore-1 TTA CCC CAT GCC CAG CT Quencher-1 WT Probe
45121 Fluorophore-2 TGC ATG ACT CAG CTC CCA TAC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45117 0.40 uM
45118 0.40 uM
45119 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.