Ptc 200 thermocycler

As shown in Figs. 1A and 2A, DNA fragments containing intron 20 (S-type and L-type) were amplified by PCR using two different pairs of primers a and b, and e and f, which were located on the exon 20 and exon 21 of PolA1 gene, respectively. The primers, listed in Supplemental Table 2, were designed based on the sequence of rice PolA1 gene (NC_029261, DDBJ). Subsequently, PCR amplification was performed with ExTaq DNA polymerase (TaKaRa, Shiga, Japan) according to manufacturer’s instruction. The PCR conditions were 40 cycles of 94°C for 1 min, 58°C for 1 min for annealing, and 72°C for 2 min for elongation in a PTC200 thermocycler (MJ Research, Waltham, MA, USA).
The amplified PCR products were subjected to 1.0–1.5% agarose gel electrophoresis and purified using a PCR purification kit (QIAquick; Qiagen, CA, USA). DNA sequences of the purified PCR products were determined by direct sequencing with the same primer as used for PCR amplification in an automated DNA sequencer ABI310 (Applied Biosystems, CA, USA) with a Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, USA). Sequences of the L-type intron 20 were determined by using primers c, d1, and d2 as a sequencing primer (Supplemental Table 2). The determined intron 20 sequences of PolA1 genes in Oryza species were registered in the DDBJ as accession nos. (LC638415–LC638446).

Genomic DNA contamination was first eliminated using the RT2 First Strand Kit (Qiagen Ltd. Manchester, UK). Briefly, 400–500 ng RNA, diluted as required into a final volume of 8 µL using RNase-free water, and 2 µL Buffer GE were added to a 0.5 mL PCR tube. The samples were then heated to 42 °C for 5 min and cooled to 4 °C for 10 min in a PTC-200 Thermocycler (MJ Research). Complimentary DNA was produced from the gDNA elimination reaction using the above RT2 First Strand Kit by adding 4 µL 5× Buffer BC3, 1 µL Control P2, 2 µL Buffer RE3 and 3 µL RNase-free water. The samples were incubated at 42 °C for 15 min followed by 95 °C for 5 min in the thermocycler and the produced cDNA produced frozen at −20 °C. For analysis of steady-state mRNA levels, relative abundance of transcripts of interest was assessed by quantitative-PCR in RT2 SYBRgreen Mastermix (Qiagen Ltd. Manchester, UK) with a StepOnePlus detection system (Applied Biosciences). RT2 primer assays for rat Sod2, Gss, Gstk1, Lonp1, Tnf and Il1α were obtained from QIAgen. Thermocycler parameters were as follows: initial incubation, 95 °C for 10 min; with 40 cycles of both elongation, 15 s at 95 °C; and cooling, 1 min at 60 °C. Expression levels were normalised to Rn18s using the ∆∆CT method, and subsequently to samples of the control group to give fold-changes.

Sex determination of two human fetuses (Gestational weeks 11: GW11) was obtained by isolating DNA from extracted tissues using the NucleoSpin Tissue Kit (Macherey-Nagel), according to manufacturer instructions, and the extracted DNA was stored at −20°C until use. The DNA concentration (absorbance at 260 nm) and purity (A260/A280 ratio) were assessed using the NanoDrop 1000 Spectrophotometer (ThermoScientific). A PCR was performed in a PTC-200 thermocycler (MJ Research) using the following steps: 94°C for 3 min and 35 cycles of 94°C for 1 min; 56°C for 30 s; 72°C for 30 s and 72°C for 5 min. For genotyping the following primers were used: SRY sense 5’-AGCGATGATTACAGTCCAGC-3’ and antisense 5’-CCTACAGCTTTGTCCAGTGG-3’; FGF16 sense 5’-CGGGAGGGATACAGGACTAAAC-3’ and antisense 5’-CTGTAGGTAGCATCTGTGGC-3’. The presence of DNA extracted from the two sexual chromosome X (FGF16: 495 bp) and Y (SRY: 538 bp) was assessed by electrophoresis on a 2% agarose gel.
The DNA was visualized thank to SybrGreen staining under an UV transilluminator (Biorad Gel Doc XR + with Image Lab Solfware) and compared against a known molecular weight marker (DNA Step Ladder 50 bp, Promega).