Qiaamp dna mini kit 250

Transgenic animals and control littermates were genotyped from ear cuts. First, a DNA extraction was performed (QIAamp DNA Mini Kit (250), QIAGEN Benelux B.V., Belgium), followed by two separate PCR reaction with Extract-N-Amp™ PCR ReadyMix (Sigma-Aldricht, Belgium) and specific primers (all from Invitrogen, ThermoFisher Scientific, Belgium) as described previously [29 (link)]. The PCR reaction mixture was incubated at 94 °C for 3 min, then 40 cycles of amplification (94 °C, 30 s; 60 °C (tTA) or 55 °C (APP), 1 min; 72 °C, 1 min), and finally elongation at 72 °C for 4 min.

Tail snip samples were collected from pups before P3. DNA was extracted using the reagents and protocol of the QIAGEN QIAamp DNA Mini Kit 250 (catalog 51306). After extraction, PCR was performed using 0.25 μL mixture of primers 5′ AGA AAA GTT CTT CAC TGG TTG ACA 3′ (forward) and 5′ CAT CAT CAT CCC TGG TTC CTG 3′ (reverse), 6.25 μL Quanta Bio’s AccuStart GelTrack PCR SuperMix (catalog 95136), and 4 μL DDI H₂O. The reaction was run at 95°C for 2 minutes, followed by 35 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 minute, followed by 72°C for 1 minute. After completion of PCR, excess nucleotides were removed using 4 μL of applied biosystems ExoSAP-IT Express PCR Product Cleanup (catalog 750011). The reaction was run at 37°C for 1 minute, followed by 37°C for 14 minutes, and ending with 80°C for 15 minutes. Samples were sent to Eton Biosciences with the forward primer for sequencing to analyze the C1186T loci of exon 12 of the Tmem67 gene.

The whole cell and cytosolic DNA was purified by QIAamp DNA Mini Kit (250) LN:154041343 (Qiagen, Valencia, CA). Quantitative real-time PCR was done using Lightcycler 480 system (Roche Life Science, Indianapolis, IN) and analysis was performed using the Roche analysis software. The thermo-cycler conditions were 95 °C for 5 min, 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, totalling 50 cycles. The primer sequences used to amplify human D-loop were: sense 5′-CATCTGGTTCCTACTTCAGGG-3′ and antisense 5′-CCGTGAGTGGTTAATAGG GTG-3′.

Blood was drawn in the morning on the first day of medical in-processing and before any physical training for the candidates. It was collected in EDTA-containing tubes and stored on ice for transport (~1 hour). Upon arrival, it was immediately centrifuged for buffy coat isolation. Plasma was aliquoted for protein analysis and stored at −80°C until being ready for use. Buffy coats were transferred to 1.5 mL centrifuge tubes and stored (<48 hours) at 4°C until further processing.
DNA was extracted from the buffy coat using the QIAamp DNA mini kit 250 as per package protocol (Catalog # 51106 Qiagen, Valencia, CA, USA). DNA was harvested from 455 samples. DNA concentrations were measured via spectrophotometry at 260 nm (NanoDrop ND-1000, ThermoFisher Scientific, Wilmington, DE). DNA purity was assessed by 260/280 nm; ratios within 1.80 to 1.85 were considered acceptable. Samples out of tolerance were reextracted as described above. Portions of DNA stock solutions were subsequently diluted to 25 ng/μL to create standardized working stock solutions.

Strain DNA was extracted using the QIAamp® DNA Mini Kit (250) (QIAGEN) following the manufacturer’s instructions. DNA was eluted in 100 μl of elution buffer and stored at 4 °C.

Twenty cultured T. vaginalis samples previously collected from male and female patients attending the OCD were investigated for the presence of symbiotic Mycoplasma species. The vaginal and urethral swabs were smeared onto T. vaginalis specific agar plates [13 (link)]. After microscopic observation for the presence of T. vaginalis at the OCD, positive samples were transferred to the ISPTM. Subsequently, the samples were suspended into liquid TYM Medium (Trypticase-Peptone Medium) and cultured microaerobically at 37 °C. The QIAamp^® DNA Mini Kit 250 (QIAGEN, Hilden, Germany) was used to isolate DNA from the pure T. vaginalis cultures. To detect the intracellular/membrane-associated genital mycoplasmas, the MGSO/GPO-1 primers were used, and the PCR products were visualized and subsequently sequenced as described.