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Tph2 Mouse Genotyping by PCR

PCR Success Story #7

Project Description

This project consisted in optimizing Tph2 Mouse Genotyping by PCR protocol for a well established lab located at the University of Ottawa. The customer had difficulties genotyping their Tg(Tph2-cre/ERT2)6Gloss mice from Jackson Labs. They were using Taq DNA Polymerase from ”BasicBio”. Cost was an issue, but performance was critical. The researcher provided us with PCR primers 11679 + 12523 for transgene detection and oIMR8744 + oIMR8745 as an internal control, 2 hemizygous mice gDNA samples and mice gDNA samples that are negative for the TPH2 transgene. Please find bellow what we have accomplished using Transgen Biotech’s EasyTaq and TransTaq HiFi DNA polymerases.

Project Details

Client: University of Ottawa
Date: April 29th, 2016
Type of experiment: Tph2 Mouse Genotyping by PCR
DNA Polymerase: EasyTaq and TransTaq HiFi DNA Polymerase.

Competitor: none

Tph2 Mouse Genotyping Protocol JAckson lab Tg(Tph2-cre/ERT2)6Gloss
Tph2 Mouse Genotyping JAckson lab figure Tg(Tph2-cre/ERT2)6Gloss

Tph2 Mouse Genotyping

We’ve shortened the PCR run time by ∼30 min as compared to the Tph2 Mouse Genotyping PCR protocol the researcher was using. This allowed the researcher to save time on their routine genotyping experiments.

Complicated PCR protocol… Genotyping should be EASY

Tph2 Mouse Genotyping with EasyTaq and TransTaq HiFi

EasyTaq and TransTaq HiFi work perfectly

Using a much shorter PCR cycling protocol than suggested by Jackson labs for Tph2 Mouse Genotyping and combining 4 primers for ”control” or ”transgene” allele detection, both EasyTaq and TransTaq HiFi (a hot start DNA polymerase) successfully amplified the transgene and control bands.

This assay does not distinguish hemizygous from homozygous transgenic animals.

Transgene ~300 bp

Internal Positive Control = 200 bp

Tph2 Mouse Genotyping

PCR reaction setup for Tph2 Mouse Genotyping:

  • H2O : 14.6 ul
  • 10x buffer : 2 ul
  • dNTPs (2.5mM): 1.6 ul (0.2 mM final)
  • F1 (10 uM): 0.4 ul (200 nM final)
  • F2 : 0.4 ul
  • R1 : 0.4 ul
  • R2 : 0.4 ul
  • gDNA : 0.4 ul unpurified gDNA
  • Polymerase (5 u/ul) : 0.2 ul

PCR cycling for Tph2 Mouse Genotyping

Denaturation: 60s at 94 °C

35 x

  1. Denaturation: 15s at 94 °C
  2. Annealing: 20s at 57 °C
  3. Extension: 10s at 72 °C

Final extension: 120s at 72 °C

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