DsRed Mouse Genotyping by PCR
PCR Success Story #3Project Description
DsRed Mouse Genotyping by PCR
This small challenge consisted in optimizing the DsRed Mouse Genotyping PCR protocol for a well established lab located at the Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CR-CHUM). The customer contacted us by saying that they offered us an opportunity to prove ourselves to their lab - I always see that as a challenge! They were using Platinum Taq HiFi from Invitrogen for Tg(Cspg4-DsRed.T1)1Akik mouse genotyping by PCR. Their PCR protocol for DsRed Mouse Genotyping was poorly designed and was copied from Jackson Laboratories’ website. Cost was an issue, but performance was critical. The researcher provided us with PCR primers (oIMR7338, oIMR7339, oIMR8699 and oIMR8700), 1 hemizygous control and 2 of their mice gDNA samples to test. Hence, you will find bellow what we have accomplished using TransBionovo’s TransTaq HiFi DNA polymerase.
What does your Taq supplier do for you?! ?
Project Details
Client: CR-CHUM
Date: April 15th, 2016
Type of experiment: DsRed Mouse Genotyping by PCR
DNA Polymerase: TransTaq HiFi DNA Polymerase.
Competitor: Invitrogen Platinum Taq HiFi
Too complicated, bad results, long PCR run... Genotyping should be EASY
We didn’t like their protocol so we did not even give it a try…
DsRed Mouse Genotyping with TransTaq HiFi
TransTaq HiFi worked perfectly for DsRed Mouse Genotyping
Using a much shorter PCR cycling protocol than what suggested by Jackson labs. DsRed Mouse Genotyping was performed by combining all 4 primers for WT or transgene allele detection, TransTaq HiFi (a hot start DNA polymerase), but not Platinum Taq HiFi DNA polymerase, successfully amplified both mutant and transgene bands.
PCR reaction setup for DsRed Mouse Genotyping
- H2O : 15.2 ul
- 10x buffer : 2 ul
- dNTPs (2.5 mM): 1.6 ul (0.2 mM final)
- F1 (20 uM): 0.2 ul (200 nM final)
- F2 : 0.2 ul
- R1 : 0.2 ul
- R2 : 0.2 ul
- gDNA : 0.2 ul unpurified gDNA
- Pol (5 u/ul) : 0.2 ul
PCR cycling DsRed Mouse Genotyping
Denaturation: 60s at 94 °C
35 x
- Denaturation: 15s at 94 °C
- Annealing: 15s at 55 °C
- Extension: 10s at 72 °C
Final extension: 120s at 72 °C
DsRed Mouse Genotyping Comparison with other Taq DNA Polymerases
We’ve compared other competitor Taq DNA polymerases to EasyTaq and TransTaq HiFi (hot start) in DsRed Mouse Genotyping by PCR. The Negiaq Taq DNA Polymerase was better at amplifying both alleles than the BEN Taq. Unike TransTaq HiFi, Negiaq Taq DNA Polymerase is not a hot start DNA polymerase and its cost is about 580$ per 1000u.
Want perfect PCR too?
Put us in charge of optimizing PCR conditions for you. It’s FREE !
DNA Polymerase Comparison Chart
Specifications and pricing of commercially available Taq DNA Polymerases and High-Fidelity DNA Polymerases in Canada.
Detection Limit of High-Fidelity DNA Polymerases on gDNA
Published on Jan 30, 2017 by Simon Roy, Ph.D Detection Limit of High-Fidelity DNA Polymerases using Human gDNA for Template We previously determined the detection limit of different Taq DNA Polymerases using human genomic DNA as a template for PCR. For this instance,...
PCR Purification Kit Comparison - DNA Clean Up
Which is the Best Kit for DNA Clean Up? Our aim was to perform a PCR Purification Kit Comparison from different vendors. At Civic Bioscience, we distribute the Favorprep™ and EasyPure® product lines from FAVORGEN and TransBionovo respectively. First, the FavorPrep™...
miniPCR Review : How I use a portable PCR thermocycler to help life scientists day-to-day.
The miniPCR and blueGel systems represent the core instruments involved in the assistance we provide to real scientists who conduct life science research in Canada. Both laboratory instruments were essential in solving dozens of problems that prospect customers were...