Protocol 44146: Probe Assay - Grid1<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

  
>chr14:35026435+35026539 105bp AACTGCCAGAACTCAGAGCAC GGTTGTCTGGTAGCATCAGG

Mutant=  111 bp

Wild Type = 105 bp

Sequence

Wt Sequence (deletions in lower case):

AACTGCCAGAACTCAGAGCACATGGGAGAGTATAGAGCTTGTTACAAAGGGTGATTCTGGCatagcttgggtggcagaaagggttcctgatgctaccagacaaccaccttatatcctggggctggatactgatgagggatactgggtctatgttgctgccttgcaccatgctctgaataagtgaagtagatggggactcttgttacttctgatggttgcgtgagttgggtgtgttggcgttagttgccctctcagatagatcctttcccacttaaaaaaatgattctttcccctcttcttccatgctcagatatccgtgggctccaaagcttcctggatcaggcctcaaggctgggtctggatgtctctttacaaaaggtggacaagaacatcagtcacgtgttcactagccttttcaccaccatgaagaccgaggagctgaatcgctaccgggacacactgcgccgtgccattctactgctcagcccacagggggcgcactccttcatcaatgaggtaggtcatgggatctgaggccattcatgcctgatgctcaggactgctgaatggcctaagtgtgtaccagaaacaacagagaacagagggctcagcccacagcacctaaccattcaggaagaggcctgaatgtgacttggatactggtttgtcctcagcagatgactcggttccttcacagaccacgaggaggctttgaggctggtgagaagcctgcATATGTTACTGCCTCAATTCTCTGAGTTGAAACCCAGCATTTTTATTCCC

 

This mutation is a 672 bp deletion beginning at Chromosome 14 position 35,026,496 bp and ending after 35,027,167 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64872 AAC TGC CAG AAC TCA GAG CAC Common A
64873 GGT TGT CTG GTA GCA TCA GG Wild type Reverse A
64874 GGG AAT AAA AAT GCT GGG TTT C Mutant Reverse A
64875 Fluorophore-1 ATA GCT TGG GTG GCA GAA AGG Quencher-1 WT Probe
64876 Fluorophore-2 TGA TTC TGG CAT ATG TTA CTG CCT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64872 0.40 uM
64873 0.40 uM
64874 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.