Protocol 40430: Probe Assay - Tg(Ins2-TFRC/OVA)296Wehi-Chr3
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

Mutant = 94 bp
Heterozygote = 93 bp and 94 bp
Wild type = 93 bp

>chr3:98061028+98061120 93bp ATGTGGACCCTTGCATCTTG CCTATGTGCCAAACACAGTGAC

This assay is capable of distinguishing hemi from hom.  Transgene insertion site is known to be on mouse Chr 3.

This assay is NOT able to be used for copy number evaluation.  If this is required, it is suggested to type by qPCR.

This protocol can be used for conventional PCR.  Omit probes and run as a separated assay.

Sequence

Wt Sequence:

AATTGAAGTCTGGAGCCTTTAAGTTGTAAAGATTATGTGGACCCTTGCATCTTGCCCTTTGGCATCCGTTAAGAATTTGAACAGCTGAATGGCTTCCATATGCTAGTCACTGTGTTTGGCACATAGGGTATAGTAGAGAATAACTCTTGTCTATTAGAGAAGATAAATAATTGTAGTA

Mutant Sequence:

CCCAGTGGTTCCCAAACTTCCAGTGTAATTGAAGTCTGGAGCCTTTAAGTTGTAAAGATTATGTGGACCCTTGCATCTTGCCCTTTGGCTGCATTTTTCCCCCATAAACCTTCTGCTGATGTATCAGGTTGCATTTTTCAGTGTGATGACTCCAATTTCATTCCCTTAAGCCTTCATCCATGAGTTACCAGTATTACTCTGTGTAAAGGAAAAGTGAATTGCACCTGTT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
55827 ATG TGG ACC CTT GCA TCT TG Common A
55828 CCT ATG TGC CAA ACA CAG TGA C Wild type Reverse A
55829 Fluorophore-1 CAT CCG TTA AGA ATT TGA ACA GCT Quencher-1 WT Probe
55830 TGG AGT CAT CAC ACT GAA AAA TG Mutant Reverse A
55831 Fluorophore-2 CTG CAT TTT TCC CCC ATA AAC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
55827 0.40 uM
55828 0.40 uM
55830 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.