Taq DNA Polymerase Comparison (Part 2)

Taq DNA Polymerase Comparison…which one should you choose?

Nowadays, many suppliers offer Taq DNA Polymerases. Our aim was to perform a Taq DNA Polymerase Comparison. Our Taq DNA Polymerase Comparison assay consisted in PCR amplification of a fragment of the human β-globin gene from purified human genomic DNA or mouse genotyping of DsRED, Cre or flox/src from unpurified mouse gDNA. PCR and DNA electrophoresis were performed with the miniPCR and blueGel systems respectively.

PCR reaction setup for β-globin amplification:

• 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 (200 nM final)
• Reverse primer (10 uM) : 0.4 ul (200 nM final)
• gDNA 10 ng/ul : 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 amplification :

1. Denaturation: 60s at 94 °C
2. 35 x
   • Denaturation: 15s at 94 °C
   • Annealing: 15s at 59 °C
   • Extension: 35s at 68 °C
3. Final extension: 300s at 68 °C

Which Taq should you choose for mouse genotyping by PCR?

This assay consists in PCR amplification of DsRED, Cre or flox/src from unpurified mouse gDNA..

PCR reaction setup for DsRed amplification:

• H2O : to 20 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 : 1 ul unpurified gDNA
• Polymerase (5 u/ul) : 0.2 ul

PCR cycling for DsRED amplification :

1. Denaturation: 60s at 94 °C
2. 35 x
   1. Denaturation: 15s at 94 °C
   2. Annealing: 20s at 57 °C
   3. Extension: 10s at 72 °C
3. Final extension: 300s at 72 °C

PCR reaction setup for Cre and flox/src amplification:

• H2O : to 20 ul
• 10x buffer : 2 ul
• dNTPs (2.5mM): 1.6 ul (0.2 mM final)
• F1 (10 uM): 0.4 ul (200 nM final)
• R1 : 0.4 ul
• gDNA : 1 ul unpurified gDNA
• Polymerase (5 u/ul) : 0.2 ul