Furthermore, the HotStarTaq polymerase present in the QuantiTect Multiplex PCR buffer in the PCR master mix is inactive at 30°C.
For the 50 μL PCR reaction, 25 μL 2× PCR buffer, 0.6 μL 2× primers (25 pmol μL−1), 0.3 μL probe (25 pmol μL−1), 1 μL cDNA, and 22.8 μL DEPC water (Sigma) were mixed together.
The PCR mixture (25 µL) contained 1× PCR buffer (MgCl2+), 2.5 mM dNTPs mix, 0.5 µM of each primer, and 1 U of Taq DNA polymerase (Takara, Bio Inc, Shiga, Japan).
PCR was performed with Ex Taq polymerase (Takara) in a PCR buffer.
These PCR product substrates were incubated with 0.2 units nuclease in a common PCR buffer for 5 minutes at room-temperature.
Each PCR reaction was carried out in final volume of 25 μL, containing 10 ng of first-strand cDNA, 2.5 mM MgCl2, 10 μM PCR primers, 2 mM dNTP and 0.5 U Taq DNA polymerase in 10 × PCR buffer.
The standard PCR reaction mix (25 μl) consisted of 100 ng cosmid DNA, 0.2 mM of each primer, 2.5 μl of 10X PCR buffer, 1 μl of 10 mM dNTPs mix, and 1.25 U of Pfu DNA polymerase (Fermentas, USA).
Rt-PCR was performed with Platinium taq (Invitrogen), 1.5 mM MgCl2, 200 nM dNTPs, 200 nM each primer, 1x PCR buffer.
In both cases, we used a 10 μM concentration of target DNA in 1× PCR buffer.
The 20-μL PCR solution contained 1× PCR buffer, 200 μM of each dNTP, 0.25 μM of each primer, 1.5 μL of supernatant (template DNA), and 1 unit of Taq DNA polymerase.
PCR reactions were carried out in a 10 μl reaction containing 2 μl of first strand cDNA, 1X PCR buffer, 125 μM dNTPs, 1.5 mM MgCl2, 0.2 μM primers and 1U Taq polymerase.
