Silverstar taq dna polymerase

Fetal gonads were fixed in 4% paraformaldehyde (PFA) (MERCK, Darmstadt, Germany) overnight (o/n) at 4°C and adult ovarian pieces were fixed either in Bouin’s solution (Sigma-Aldrich, St. Louis, USA) or 4% PFA o/n at 4°C. Thereafter, the tissues were washed 3× in phosphate-buffered saline (PBS), transferred to 70% ethanol solution and embedded in paraffin using a Shandon Excelsior tissue processor (Thermo Scientific, Altrincham, UK). The material was sectioned (5 μm) using a RM2065 microtome (Leica Instruments GmbH, Wetzlar, Germany) and mounted using Prolong Gold (Life Technologies, Carlsbad, USA) on StarFrost slides (Waldemar Knittel, Braunschweig, Germany). The sex of the human fetal gonads was determined using a genomic PCR for AMELOGENIN (FW 5′ CTG ATG GTT GGC CTC AAG CCT GTG 3′ and RV 5′-TAA AGA GAT TCA TTA ACT TGA CTG-3′), that resulted in two different sized amplicons when from the X (977 bp) or Y (790 bp) chromosomes [28 (link)]. The PCR was performed using SilverStar Taq DNA polymerase (Eurogentec, Seraing, Belgium) with a PCR cycle of 95°C for 5 minutes, 34 times 95°C for 1 minutes, 60°C for 30 seconds, 72°C for 2 minutes and a final extension step at 72°C for 10 minutes. The PCR products were run on a 1.5% agarose gel.

DNA was extracted using the UltraClean^TM Soil DNA isolation kit (MoBio Laboratories, Inc., Carlsbad, CA, United States) and amplified by PCR using the 16S rRNA gene universal eubacterial primers E338F (5′-ACTCCTACGGGAGGCAGCAGT-3′) and E797R (5′-GGGTATCTAATCCTG-3′) (amplicon length of 459-bp covering the variable V3 region of the 16S rRNA gene). The PCR mixtures were prepared with 2.5 μl 10x PCR buffer (Eurogentec, Seraing, Belgium), 1 μl MgCl₂ (50 mM; Eurogentec), 2.5 μl dNTPs (2 mM; Thermo Scientific, Waltham, MA, United States), 1.0 μl of each primer (20 pmol μl^-1 each; Eurogentec), 0.2 μl Silverstar Taq DNA polymerase (5.0 U μl^-1; Eurogentec) and 1 μl standardized template DNA (10.74 ng μl^-1) in a total reaction volume of 25 μL. The PCR was performed with the follow temperature/time conditions: initial denaturation at 94°C for 2 min, 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, and a final extension for 5 min (72°C). PCR products were evaluated on gel electrophoresis, the bands then were excised and purification was performed using a Qiaquick Gel extraction kit (Qiagen, Venlo, Netherlands). For the quantification of the PCR products, we used the PicoGreen dsDNA Assay Kit (Life Technologies, United States). Sequencing was performed at the Genomics Core of KU Leuven (Belgium) using a Roche 454 GS FLX+ (Switzerland).