Tph2 Mouse Genotyping by PCRPCR Success Story #7
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.
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
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
- Denaturation: 15s at 94 °C
- Annealing: 20s at 57 °C
- Extension: 10s at 72 °C
Final extension: 120s at 72 °C
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