Geneamp pcr 9700

The PCRs were carried out on a Gene AMP® PCR 9700 Applied Biosystem Machine. The PCR mixture contained 10 µl of OneTaq Hot Start Quick-Load (from New England Biolabs), 0.8 µl of forward primers, 0.8 µl of reverse primers, 1 µl of DNA template from the extracted DNA, and 7.4 µl of nuclease-free water making a total volume of 20 µl. The primers used amplified the complete PfAMA1 gene. The PCR was programmed for 25 cycles.

The PCRs were carried out on a Gene AMP® PCR 9700 Applied Biosystem Machine. The PCR mixture contained 10 µl of OneTaq Hot Start Quick-Load (from New England Biolabs), 0.8 µl of forward primers, 0.8 µl of reverse primers, 1 µl of DNA template obtained from the product of the first round of PCR, and 7.4 µl of nuclease-free water making a total volume of 20 µl. The primers used amplified Domain I of PfAMA1 where majority of genetic diversity occurred. The PCR was programmed for 30 cycles. Cycling conditions for both PCRs were as follows: initial denaturation at 95°C for 5 minutes, followed by 25 cycles (first round) or 30 cycles (second round) of denaturation at 94°C for 1 minute, annealing at 58°C for 2 minutes, and extension at 72°C for 2 minutes. The final extension was carried out at 72°C for 5 minutes and held at 5°C. The negative control's PCR settings were the same as those used in the experiment, but it did not contain any of the genomic DNA that was extracted from Plasmodium falciparum. The primers used are shown in Table 1.

Placental tissues were immediately cut into small pieces and snap‐frozen in liquid nitrogen and then stored at −80°C. Isolation of total RNA from the placenta and BeWo cells; a trophoblast‐derived epithelial cancer cell line was performed with ISOGEN (NIPPON GENE, Tokyo, Japan) according to the product manual. After extracting RNA, the samples were treated with DNase to remove genomic DNA. RNA was purified by extraction with phenol, chloroform, and isoamyl alcohol and ethanol precipitation. Reverse transcription was performed with Superscript III First‐Strand Synthesis System (Life Technologies) according to the product manual. PCR reaction was performed with two or three kinds of primer pairs for each transporter (Table 1) using Gene Amp PCR 9700 (Life Technologies).

Twenty‐four Y‐STR loci were amplified simultaneously using DNATyper^TM Y24 amplification kit (Physical evidence identification center) in a GeneAmp^® PCR 9700 (Life Technologies) thermal cycler, according to the manufacturer's recommendations. Subsequently, separation and detection were performed using an Applied Biosystems™ 3500 Series Genetic Analyzer (Life Technologies). Finally, the raw data were analyzed using GeneMapper ID v4.1 software (Life Technologies). We strictly followed the recommendations of the DNA Commission of the International Society of Forensic Genetics on the analysis of Y‐STRs (Gusmao et al., 2006).

Genomic DNA was extracted using Chelex-100 [11 (link)]. PCR amplification was performed using AmpFISTR^® Yfiler^® Plus and Identifiler^TM PCR amplification kits (Thermo Fisher Scientific, CA, USA) in a GeneAmp^® PCR 9700 (Thermo Fisher Scientific, CA, USA) thermal cycler, according to respective manufacturer specifications. The AmpFISTR^® Yfiler^® Plus amplification kit (Thermo Fisher Scientific, Waltham, MA, USA) can co-amplify 25 Y-STR loci with six dyes, including seven rapidly mutating loci [12 (link)]. The AmpFISTR^® Identifiler^TM PCR Amplification kit (Thermo Fisher Scientific) can co-amplify 15 autosomal STR loci and the Amelogenin locus with five dyes. Fragments of the 25 Y-chromosomal and 15 autosomal STR loci were produced simultaneously. Separation and detection of amplicons was performed on an Applied Biosystems™ 3500 Series Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). Data were analyzed using GeneMapper ID v4.1 software (Thermo Fisher Scientific, Waltham, MA, USA). Control DNA 007 was included as a standard reference in each batch of genotyping. We strictly followed the recommendations of the DNA Commission of the International Society of Forensic Genetics (ISFG) for Y-STR analysis [13 (link)].

Total RNA was extracted from NB cell lines using RNeasy Mini Kit (Qiagen, Hilden, Germany). The cDNA was reverse-transcribed from total RNA using SuperScript II reverse transcriptase and random primers (Thermo Fisher Scientific) and subjected to PCR-based amplification using rTaq DNA polymerase (TaKaRa, Shiga, Japan). The primer sets used for PCR were as follows:
BMCC1 forward: 5′- GAAGCCTCTGGTCCAGTCAG-3′;
BMCC1 reverse: 5′-CTTCGGCCGTATATTCTGGA-3′;
E2F1 forward: 5′-TGCAGAGCAGATGGTTATGG-3′;
E2F1 reverse: 5′-GTTCTTGCTCCAGGCTGAGT-3′;
TAp73 forward: 5′-CCTCTGGAGCTCTCTGGAAC-3′;
TAp73 reverse: 5′-GAAGACGTCCATGCTGGAAT-3′;
GAPDH forward: 5′-ACCACAGTCCATGCCATCAC-3′;
GAPDH reverse: 5′-TCCACCACCCTGTTGCTGTA-3′.
A GeneAmp PCR 9700 and a Veriti thermal cycler (Thermo Fisher Scientific) were used for PCR, and the amplified DNA fragments were separated using agarose gel electrophoresis. Then, they were stained with ethidium bromide and detected with a UV-transilluminator (ATTO, Tokyo, Japan). The expression levels of BMCC1 in SK-N-AS cells were measured by quantitative real-time PCR (Q-PCR) using ABI PRISM 7500 system (Thermo Fisher Scientific). TaqMan probes for BMCC1 (Hs00322421_m1) and GAPDH (4310884E) were purchased from Thermo Fisher Scientific.