Hard shell 96 well pcr plate

Differential scanning fluorimetry experiments were carried out using a CFX Connect RT-PCR detection system (Bio-Rad) and using Hard-Shell 96-well PCR plates (Bio-Rad), which are compatible with the excitation and emission wavelength of SYPRO orange. The temperature range was 4 to 95°C with an increment of 1°C every 45 s. The fluorescence was measured every 15 s. Data were analyzed using GraphPad Prism 6.

A published protocol was followed without modifications^{20 (link)}. For 2 × master mix, the final concentrations of components were the following: 2 × Q5 reaction buffer, 0.5 µM primer and 0.5 µM 197-nt template, 0.1 mM dNTPs (except for target dNTP) and 2.5 µM EvaGreen (Biotum). Q5 DNA polymerase (NEB) was then added to reaction mixtures as follows: final concentration of 20 U/ml for dATP and dTTP, and 10 U/ml for dGTP and dCTP detection. Five µl of each 2 × master mix was pipetted into Hard-Shell® 96-Well PCR Plates (Bio-Rad) according to dNTP species. Then 5 µl of samples was added to each well for dNTP quantification by real-time PCR. For initial optimization runs, the thermal cycler (CFX96, Bio-Rad) was programmed to heat the plate to 98 °C (10 s) followed by cooling to the final reaction temperature (67 °C). The baseline fluorescence was immediately read after reaching the target temperature. Thereafter, the fluorescence (SYBR Green/FAM channel of the instrument) was recorded typically once every 5 min for total of 75 min. Data were normalized to the volume of estimated samples.

Total DNA from cultured cells was isolated using a NucleoSpin Tissue Purification Kit (Macherey-Nagel; 740952) according to the manufacturer's protocol. The mtDNA content was quantified and normalized to nuclear DNA (nucDNA). Measurements were performed using quantitative real-time PCR (qPCR) with primers for cytochrome b (CytB, mtDNA) and amyloid precursor protein (APP, nucDNA): CytB-Fw 5′-GCCTGCCTGATCCTCCAAAT-3′, CytB-Rv 5′-AAGGTAGCGGATGATTCAGCC-3′; APP-Fw 5′-TTTTTGTGTGCTCTCCCAGGTCT-3′, APP-Rv 5′-TGGTCACTGGTTGGTTGGC-3′. Each qPCR reaction contained 25 ng of purified total DNA, 2.5 mM of forward and reverse primers, 10 μl of 2× SYBR Green Master Mix (Bio-Rad; #4309155) and was measured in triplicate in Hard-Shell 96-Well PCR plates (Bio-Rad; #HSP9635). The amplification program was as follows: 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The fluorescent signal was acquired by a CFX96 Real-Time System (Bio-Rad). The absence of non-specific amplicons was confirmed using melting curve analysis. Fold changes in the relative mtDNA copy number after C17orf80 depletion or ddC treatment were calculated using the 2^−ΔΔCT method.

qPCR was performed using a CFX96 Connect apparatus (Bio-Rad, Japan). Hard-Shell® 96-Well PCR Plates (Cat # HSP 9601, Bio-Rad) sealed with optically clear adhesive seals (Microseal® ‘B’ seal, Cat # MSB1001, Bio-Rad) were used in all experiments. The thermocycler program consisted of an initial hot start cycle at 95 °C for 3 min, followed by 33 cycles at 95 °C for 10 s and 59 °C for 30 s. Mouse actin beta (Actb) was amplified using the following primers: F-5′-AACCCTAAGGCCAACCGTGAA-3′, R-5′-ATGGCGTGAGGGAGAGCATA-3′ (with estimated product length 194 bp). The primers were used at a concentration of 300 nM. SYBR Green-based PCR supermix (Bio-Rad) was used for all reactions according to manufacturer’s instructions. Each reaction was performed in a final volume of 8 μL. To confirm product specificity, a melting curve analysis was performed after each amplification, and agarose gel analysis was performed to ensure the amplification of the right product (Additional file 1: Figure S1).