When a new sample - containing a value for each processed gestural parameter - comes in, the system needs a temporal reference indicating where to store the sample in the template buffer on the Time axis.
500 ng of genomic DNA was amplified in a 50 μL-reaction volume using 2.75 mM Mg2+, 500 μM of each dNTP, 2 U of Expand Long Template PCR System (Expand Long Template Buffer 2; Roche Diagnostics), and 300 nM of each primer.
LR-PCR amplifications were performed in a total volume of 50 μl of the mixture containing 300 ng of genomic DNA, 3.75 U of Expand long template enzyme mix (Roche Applied Science).), 1× Expand long template buffer 1 with 1.75 mM MgCl2, 350 μM dNTP and 15 pmol of each primer.
For a final volume of 50 μL, the PCR mixture contained 20 ng of DNA, 1x Expand Long Template buffer 3 with 2.75 mM MgCl2 (Roche), 3.2 mM (each) deoxynucleoside triphosphates, 0.4 mM of each primer, and 3.75U of Expand Long Template enzyme mix (Roche).
Reactions were carried out in 15 μl; each primer was at 1.3 μM final concentration in reactions containing template, buffer (40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgCl2, 3.75 μg ml-1 BSA, 200 μM dNTPs), 5% DMSO, 1.25 μM EvaGreen (Biotium Inc., Hayward, CA) and 0.2X Titanium Taq polymerase (BD Biosciences, Palo Alto, CA).
Reactions were carried out in 20 μl; each primer was at 0.5 μM final concentration in reactions containing template, buffer (40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgCl2, 3.75 μg ml-1 BSA, 200 μM dNTPs), 5% DMSO, 0.25× SYBR Green I, and 0.2× Titanium Taq polymerase (BD Biosciences, Palo Alto, CA).
Sharing the same template and buffer by these two enzymes largely simplifies experimental procedures.
The reaction mixture with a final volume of 50 μl had 100 ng of DNA template, 1× buffer (Tris-HCl 100 mM, KCl 500 mM), 3.0 mM of MgCl2, 0.2 mM of each dNTP, 0.3 μM of each primer, and 1 U of Taq polymerase.
25µl PCR reactions were set up containing 2µl of template, 2.5µl 10× buffer (Roche Applied Biosystems), and final concentrations of 1.5 mM Mg, 6% DMSO, 0.5 µM primers, 0.8 mM total dNTP (0.2 mM each) and 1U HotStart Polymerase (Roche Applied Biosystems).
RNAPII was phosphorylated in transcription reactions (20 µl) containing 50 µg of HeLa nuclear extract, 0.5 mM ATP, CTP, UTP and GTP, 0.2 µg of JK2 [41] linearized template in buffer C (20 mM HEPES, pH 7.9, 50 mM KCl, 6.25 mM MgCl2, 0.5 mM EDTA, 2 mM DTT and 10% glycerol).
EP-PCR reactions were carried out under the following conditions, hereafter referred to as standard: 10 ng of DNA template, 10×PCR buffer (Mg2+ Free, TaKaRa), 10 mM of each dNTP (dATP ∶ dTTP = dGTP ∶ dCTP = 1 ∶ 5), 10 µM of both primers (VHForNcoI and VLRevHindIII), 25 mM MgCl2 and 2.5 U Taq DNA polymerase (TaKaRa) in 50 µl volume.
