Amplified DNA was re-suspended in water at 37°C for 1 hour and the concentration was measured by NanoDrop (NanoDrop Technologies, Wilmington, DE). 10% by volume of the amplification products were analyzed on 0.5% alkaline agarose gels stained with SYBR-green II (Molecular Probes, Eugene, OR).
The DNA was precipitated again [ 41] and resuspended in TE to the starting volume of the DNA amplification reaction.
A volume of 20 µl of amplification reactions contained 0.4 µl cDNA, 1x Master Mix, 0.9 µM each primers and 0.25 µM probe.
The aim of the study was to describe the role of the template amount, number of amplification cycles, volume of the PCR reaction in determining the assay sensitivity and the profiling accuracy as measured by peak heights and areas.
The PCR reaction mixture contained 50 ng templates DNA, 5 pico mole of each of the primers, 200 μM dNTPs, 1 X PCR buffer (10 mM Tris HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, and 0.01 mg/ml gelatin) and 0.6 unit of Taq DNA polymerase in a volume of 20 μl and amplification of target sequences were as per earlier reports (Table 1).
The total reaction volume of a single PCR amplification was 20 μL.
The total volume of a single PCR amplification was 50 μl.
A parallel sample in which the Blepharisma was replaced with an equivalent volume of water showed no amplification.
The PCR amplification volume of 25 μL contained 10 μL of Fast SYBR Green Master Mix Life Technologiess), 5 μL of DNA, 1 μL of each 2.5 μM primer, and 8 μL of nuclease-free water.
Furthermore, as the volume of the multiple displacement amplification reaction can be scaled up, there is no theoretical limit to the amount of PCRs that could be performed on each cell.
