Protocol 35666: Probe Assay - Zfp652<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

>chr11:95752770+95752878 109bp TGGTCTTGTGCCATGAATCT CACACTAGCAGGACAAGCAGA

Mutant=  91 bp

Wild Type = 109 bp

Sequence

Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):

 AAAATAGAACATACAGAGGTTAGTGTTGGAAGATGAACTGCATGCAGGAATCGTTTTTGTTAGGATTCTGGTCTTGTGCCATGAATCTAAAGTAGATCCATTTCCTCtgccaggaggtaggatggcctcctgaagcctcagtaaataaagattctttctgcttgtcctgctagtgtgtttcctgtaacaaatccttcaagaaactctggtcccttcatgaacatatcaagattgtccatggatatgcagaaaaaaaatttgcctgtgaaatttgcgagaagaagttctataccatggctcatgtacgaaaacacatggttggtgagtctccgcccctctttctccacctgcagactcctggagtgctctctgctccacatTGGGCAGGATATGCAGAGTCAGCATACTGCAGTCAGTTTTAGCTTGTTGAGACTATATGTAGCTTGTCTTTAGTAAGCATGATCAGGTTGGACAGTGCAGTCTTCCCTACCCAGAGT

This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences GCCATCCTACCTCCTGGCAG and AGTGCTCTCTGCTCCACATT, which resulted in a 273 bp deletion beginning at Chromosome 11 position 95,752,809 bp and ending after 95,753,081 bp (GRCm38/mm10). This mutation deletes ENSMUSE00000674206 (exon 2) and 125 bp of flanking intronic sequence including the splice acceptor and donor and is predicted to cause a change of amino acid sequence after residue 299 and early truncation 54 amino acids later.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46673 TGG TCT TGT GCC ATG AAT CT Common A
46674 CAC ACT AGC AGG ACA AGC AGA Wild type Reverse A
46675 TCT CAA CAA GCT AAA ACT GAC TGC Mutant Reverse A
46676 Fluorophore-1 ATG GCC TCC TGA AGC CTC Quencher-1 WT Probe
46677 Fluorophore-2 CAT TTC CTC TGG GCA GGA TAT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46673 0.40 uM
46674 0.40 uM
46675 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.