Protocol 35543: Probe Assay - Gbp7<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

>chr3:142536213+142536308 96bp CAGAGCTATTAAGGGTGGAACC CCAGATTGAAGCCTGTGTGA

Mutant=  90 bp

Wild Type = 96 bp

Sequence

Wt Sequence (deleted region is in lowercase:

ACCTGAATAAACTCAGAGCTATTAAGGGTGGAACCTCTGCctcctgtattgtaaggattcatataaatccatggtgctttcatattctctcacacaggcttcaatctgggcaccacagttaggtctgaaactaagggcatctggatgtggtgtgtgcctcaccccagcaagcccaagttcacactcgtgcttctggacacggagggcttaggagatgtggaaaaggtaggtttggtcttggttctctgtgaaccatttgactgctgctttagtccaacttctgtgctgcttgcaagaattgcaaccaagctattgataaggagaataagttgcttcccttggtgaagtttactcctttgataaactcaaatgttctgttgtaatggaggatagtcttatcttgatacttttgtgaacaatacagttcagttcttaatatttattaagaagctgaattctagtgtttcttactatatgctacaatcctgc

This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences ATGAATCCTTACAATACAGG and CTTGCAAGCAGCACAGAAGT, which resulted in a 477 bp deletion beginning at Chromosome 3 position 142,536,240 bp where there is a 4 bp retention TGCA after 142,536,490 and then the deletion continues for 227 bp ending after 142,536,720 bp (GRCm38/mm10). This mutation deletes ENSMUSE00001000161 (exon 3) and 349 bp of flanking intronic sequence including the splice acceptor and donor and is predicted to cause an early truncation after amino acid 64.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46338 CAG AGC TAT TAA GGG TGG AAC C Common A
46339 CCA GAT TGA AGC CTG TGT GA Wild type Reverse A
46340 TCT ACT TCT ATG AAC CCA CAG CA Mutant Reverse A
46341 Fluorophore-1 TGT AAG GAT TCA TAT AAA TCC ATG GTG Quencher-1 WT Probe
46342 Fluorophore-2 AAG CAG GCA TAT ACT CTG ACA TCA G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46338 0.40 uM
46339 0.40 uM
46340 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.