Protocol 33542: Probe Assay - Zc3h13<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

>chr14:75291907+75292051 145bp TACAAGGAAGGGTTAAGGTTTG GAAAAACCACAGGAAGGTGTGT

Mut= 150 bp

Wt= 145 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

GGGTTTGGCTTAGCAAATTTTATTTTATAAAAGGAAATACAAGGAAGGGTTAAGGTTTGTAAAGTTCTCATTATATAATATTTTTGTATGTTCAATGTATACGATGTATTTCGAGCATATTAATACCCCattccaggaactactctctacctaaacacacacacaccttcctgtggtttttcattcccttatggtgttattattattggagtggcatatgcttagctggcatttgggtctctctgacttgattttgccctggtaccatcttttctccagacatttacatagtaatatttgcctcatagacacagtgtcgtaactggctgaagactggcagctgcctctatggaaacacatgtaggttcatacatggcccttcacctcgtgggaaaggctacagcagcaattacagaaggtaaactgagaactcagatattttctatagtattaaaaaccaacttttgtagtttagtgtaaaaatatgaaataaaaccatatatttggtcagattttctttttttggatagtcttattttggttgttactgttaagattaataaaactttagctttaaagtgtacatgtttacatgttttggtagtagatccatttttcaatgtgtatattcaaatcaatgaacacctgtggagagactgagtctgggaaagaacagcccttaaagagcccctACCTGACACAGCCCCAGGTGTGTATTAAATTTCTGCTTAGGCACAACTCAGACGTTTGAGAGAGTTTACTTTTCGTAAACGAGTAAAATTTAAATAAGGTTTAATTGTAATTTTCTACAGGCATTTTAAATTCATATCT

This mutation is a 563 bp deletion beginning at Chromosome 14 position 75,291,999 bp and ending after 75,292,561 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41660 TAC AAG GAA GGG TTA AGG TTT G Common A
41661 GAA AAA CCA CAG GAA GGT GTG T Wild type Reverse A
41662 CAA ACG TCT GAG TTG TGC CTA Mutant Reverse A
41663 Fluorophore-1 CCA GGA ACT ACT CTC TAC CTA AAC ACA Quencher-1 WT Probe
41664 Fluorophore-2 AAT ACC CCA CCT GAC ACA GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41660 0.40 uM
41661 0.40 uM
41662 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.