Protocol 38331: Probe Assay - Zfp513<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

>chr5:31201564-31201674 111bp AGGCCAAGATCTGGAGTTTGA CCACGACCCTTAAACACACC

Mut = 110 bp

Wt = 111 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GAGATTCCACAAGCACCCTCCCCTCTTTAGTGGATACTGAAGATTCCTTCGACgaaggtcctggggccctggtgttggagagcgatttgctactaggccaagatctggagtttgaagaggaagaggaagaggatgaaggtgacggccacaacgaccagctcATGGGCTTTGAGAGAGACTCTGAAGGTGTGTTTAAGGGTCGTGGTTTAGGATTAGTGGCAATTGGT

This mutation is a 108 bp deletion beginning at Chromosome 5 position 31,201,608 bp and ending after 31,201,715 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
53570 CCA CGA CCC TTA AAC ACA CC Common A
53571 AGG CCA AGA TCT GGA GTT TGA Wild type Forward A
53572 ACT GCT TGG GAG GGA GAT TC Mutant Forward A
53573 Fluorophore-1 AGG AAG AGG ATG AAG GTG ACG Quencher-1 WT Probe
53574 Fluorophore-2 CAC CCT CCC CTC TTT AGT GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
53570 0.40 uM
53571 0.40 uM
53572 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.