What’s the Detection Limit of Taq ? How many DNA copies from gDNA?
There are MANY Taq DNA Polymerases out there on the market. Some are driven by strong marketing and others by strong performance. Our aim was to verify the Detection Limit of Taq DNA Polymerases in PCR amplification from human genomic DNA. TransBionovo’s EasyTaq and TransStart TopTaq DNA Polymerases were compared to Taq DNA Polymerases from various vendors. Our Taq DNA Polymerase Comparison assay consisted in PCR amplification of a fragment of the human β-globin gene from purified human genomic DNA.
To determine the Detection Limit of Taq DNA Polymerases, we used either 0.02 ng, 0.2 ng, 2 ng or 20 ng of purified human gDNA as template for PCR amplification.
Details
Date: June 22th, 2016
Type of experiment: PCR from human gDNA
DNA Polymerase: EasyTaq & TransStart TopTaq DNA Polymerases
Competitors: NEB, Qiagen, BioBasic
PCR reaction setup for β-globin:
- H2O : fill to 20 ul
- 10x buffer : 2 ul
- dNTPs (2.5 mM): 1.6 ul (0.2 mM final)
- Forward primer (10 uM): 0.4 ul (0.2 uM final)
- Reverse primer (10 uM) : 0.4 ul (0.2 uM final)
- gDNA : 2 ul
- Taq DNA Polymerase (5 u/ul) : 0.2 ul
- (BioBasic Taq required the addition of MgSO4 – as recommended)
PCR cycling for β-globin :
- Denaturation: 60s at 94 °C
- 35 x
- Denaturation: 15s at 94 °C
- Annealing: 15s at 58 °C
- Extension: 30s at 72 °C
- Final extension: 120s at 72 °C
Detection Limit of Taq : EasyTaq vs TopTaq
Amplification of different fragment sizes of the human β-globin gene using EasyTaq or TransStart TopTaq DNA Polymerase.
Both EasyTaq and TopTaq were successfull at amplifying the 536 bp fragment of human β-globin from 20 ng, 2 ng and 200 pg of human genomic DNA, corresponding to 6 x 103, 6 x 102 and 6 x 101 copies of target template respectively, but not from 20 pg (6 copies).
The Detection Limit of Taq DNA Polymerases (human β-globin) were:
- EasyTaq : 200 pg –> 60 copies of template
- TopTaq : 200 pg –> 60 copies of template
Detection Limit of Taq : BEN M0267 vs Negaiq
Amplification of different fragment sizes of the human β-globin gene using BEN or Negaiq Taq DNA Polymerase.
Both BEN and Negaiq were successfull at amplifying the 536 bp fragment of human β-globin from 20 ng and 2 ng human genomic DNA but only the Negaiq Taq was able to amplify the 536 bp band using 200 pg of template (BEN Taq produced a ”ghost-like” band). Both Taq DNA Polymerases did not amplify the target using 20 pg of template. This result shows a better sensitivity of the Negaiq product as compared to the BEN Taq.
The Detection Limit of Taq DNA Polymerases (human β-globin) were:
- NEB M0267 : 0.2-2 pg –> 60-600 copies of template
- Qiagen: 200 pg –> 60 copies of template
Detection Limit of Taq : BBasic Taq
Amplification of different fragment sizes of the human β-globin gene using BBasic (Feldan) Taq DNA Polymerase.
BBasic Taq was successfull at amplifying the 536 bp fragment of human β-globin from 20 ng and 2 ng human genomic DNA but, similarly to BEN Taq, produced a ”ghost-like” band when 200 pg of template was used.
The Detection Limit of Taq DNA Polymerases (human β-globin) were:
- BioBasic Taq : 0.2-2 pg –> 60-600 copies of template
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