Protocol 43937: Probe Assay - Zfp385c<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

>chr11:100632619-100632710 92bp AACCCCTCAGAGCAAAGGTC TGGTTGAAGAGCCAGAGTCA

Mut= 91 bp

Wt= 92 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

TGAGTGAGGTGCTGTGACTTGCCTAGAGATGCAGAGCTTACATAAATGGCCAAGTTTGATTCTGAGGGCAGGCTGCCTGCATGGGAGCCCCTGTGTGAGAACAAGCTCTGTTGTAAACCTCAGGGCTCTATACATGTGGCAATGATGGTGACACTGACATCACCCCTTTGTGGCTGAGCCTTGTGTTCGTCCCTTTGACCACATATTCCTCTGGCCTATCCCCCTACTCAGCCTCCctcctagatgaggcatgagacccacgggaagaagatgacccctcccctccctcttccagaaccaggctgaggctcattacaagggccacagacacgccagaaaactcaaggctgtggaagctgccaagagcaagcagaggcctcgaaacccgaccacaaacgggacagtggtgtcttcagcctcacctccagccagtggaagccctggaacccctcagagcaaaggtcagtcttgatttcaagttcacacaacgaggCTGGCTTCTGACAGCGGCTTGAGTGACTCTGGCTCTTCAACCATCTTTCTTTCCTTTTTCTTTTGTTTGGTTTTTCAAGACAGGGTTTTTCTGTGTAGTCTCAGCTTTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTTGAACTTATAGAGATCCATCCACCTGCCTCTGCCTCATGACAGTGCTGGGATTAAAGGCATGCACCACTACTACCTGGCTTTTT

 

This mutation is a 257 bp deletion beginning at Chromosome 11 position 100,632,662 bp and ending after 100,632,918 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64400 AAC CCC TCA GAG CAA AGG TC Wild type Forward A
64401 TGG TTG AAG AGC CAG AGT CA Common A
64402 GTC CCT TTG ACC ACA TAT TC Mutant Forward A
64403 Fluorophore-1 CAA GTT CAC ACA ACG AGG CTG Quencher-1 WT Probe
64404 Fluorophore-2 CTC AGC CTC CCT GGC TTC TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64400 0.40 uM
64401 0.40 uM
64402 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.