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Cre and A2A Mouse Genotyping

PCR Success Story #6

Project Description

Re-design of Cre and A2A Mouse Genotyping

This challenge consisted re-designing the Cre and A2A Mouse Genotyping by PCR protocol for a well established lab located at the Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CR-CHUM). The researcher contacted us due to our recent successes (Story #3, Story #4, Story #5) performed for the same research center. They were using KAPA 2G Taq DNA Polymerase from ”KAPA” for Adora2atm1Masc allele mouse genotyping – Cre and A2A Mouse Genotyping. Their previous protocol was suffered from a poorly designed PCR setup and  PCR cycling protocol available from Jackson Laboratories. We also used DNA primers for amplifying Cre recombinase – the same as in Story #1). Cost was no issue, but performance was critical. The researcher provided us with PCR primers (#9057 and #9058) and many gDNA samples. Please find bellow what we have accomplished using TransBionovo’s EasyTaq DNA polymerase (Magic Taq remember?).

What does your Taq supplier do for you?! ?

Project Details

Client: CR-CHUM
Date: May 5th, 2016
Type of experiment: Cre and A2A Mouse Genotyping by PCR
DNA Polymerase: EasyTaq DNA Polymerase

Competitor: None

A2A Mouse Genotyping Jackson lab figure Adora2atm1Masc
A2A Mouse Genotyping Jackson lab figure Adora2atm1Masc PCR setup and cycling

A2A Mouse Genotyping recommendations from Jackson Labs

Too complicated, bad results, long PCR run… Genotyping should be EASY

Again, we didn’t like their protocol so we did not even give it a try… Don’t try this at home please!

A2A Mouse Genotyping is EASY

Using a much shorter PCR cycling protocol than suggested by Jackson labs for A2A Mouse Genotyping, EasyTaq easily amplified either the mutant or transgene Adora2atm1Masc bands.

A2A Mouse Genotyping with EasyTaq
Cre Mouse Genotyping with EasyTaq is also EASY with EasyTaq

Cre Mouse Genotyping with EasyTaq is also EASY

Using Cre-specific primers used in Story #1, we’ve verified the presence of the Cre transgene by PCR in the researcher’s mouse gDNA extracts. The PCR setup and cycling was exactly the same as for A2A. This should enable the researcher to save time by multiplexing A2A and Cre detection together. Nice & Easy 🙂

PCR reaction setup for Cre and A2A Mouse Genotyping

  • H2O : 15 ul
  • 10x buffer : 2 ul
  • dNTPs (2.5 mM): 1.6 ul (0.2 mM final)
  • F1 (10 uM): 0.4 ul (200 nM final)
  • R1 (10 uM): 0.4 ul
  • gDNA : 0.4 ul unpurified gDNA
  • EasyTaq (5 u/ul) : 0.2 ul

PCR cycling for Cre and A2A Mouse Genotyping

Denaturation: 120s at 94 °C

35 x

  1. Denaturation: 15s at 94 °C
  2. Annealing: 20s at 63 °C
  3. Extension: 15s at 72 °C

Final extension: 120s at 72 °C

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