Protocol 33544: Probe Assay - Zc3h7a<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

>chr16:11162786-11162895 110bp CGGAAACGCACAGTTTAAGA CCTCCACTTCCCAAGTTCTG

Mut= 101 bp

Wt= 110bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TTGAACTCAGGACCTTTAGAAGAGCAGTCAGTGCTCTTACCCACTGAGCCATCTTGCCAGCCCGAGTGTTGGATCTTTGAGGTGCCATGCGGCTTCTGGGAACTAAGTTCAGAACACCAAGCATTCCTAACTACTCAGCCATCTCTCCAGCCCCGGAAACGCACAGTTTAAGATAACTTCTGGCtgaattatgctgatagagggcctgtcatggtacagcatggtagagcgttcttgtaatctcagaacttgggaagtggaggcaagctgatctcagtaagttccaggccaatcagggttgctgccttagaactaacaaaacttttagtagaacttggtaattacaggagatttgcttcttcctctcaggcctccgctgtcatatcctgggacccaggagcagtatgcggtaaggatgtggggagccctatggctgggcacagggcctcaagagctgtaatctgcagcaggatttggcagtctagccttgactctcacacctgaccagctgccctccttcataaagagagctttactggaacaccgcggccacaggcggttgttcatgggttctctgtggtgcctgtgtgagacagggcagaactcaaatgtcccacagtgctttaaatattctctttggcatttacagaaaaagcttgccaatctcttctatatagtgtaccctttcggggcatttctggacAGTTGTCCTGCATTTTCTTTCCTGTGTTAGTGTAGTTTGAATTAGTATGCATTTTTAGTCCTTTGCCAGTTAACTAACATTTTATCAAAAATAGGTGGGATAGAATTCAGCAGAGAATGTGGGGTATCATTATTTTTGTTTTTTAAGTCCTGAAAGCAGTGAAGGAGGATTGTCTACATAGGGAG

This mutatuion is a 519 bp deletion beginning at Chromosome 16 position 11,162,346 bp and ending after 11,162,864 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41667 CGG AAA CGC ACA GTT TAA GA Common A
41668 CCT CCA CTT CCC AAG TTC TG Wild type Reverse A
41669 ACT GGC AAA GGA CTA AAA ATG C Mutant Reverse A
41670 Fluorophore-1 TGA TAG AGG GCC TGT CAT GG Quencher-1 WT Probe
41671 Fluorophore-2 CTT CTG GCA GTT GTC CTG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41667 0.40 uM
41668 0.40 uM
41669 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.