Dna extraction kit

DNA was extracted using the QIAGEN DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Subsequently, a nested PCR assay targeting a ~408 bp stretch within the S ORF was used to detect HBV DNA. First round primers were HBV_S1F 5-CTAGGACCCCTGCTCGTGTT-3, and HBV_S1R 5-CGAACCACTGAACAAATGGCACT-3, while second round primers were HBV_SNF 5-GTTGACAAGAATCCTCACAATACC-3 and HBV_SNR 5-GAGGCCCACTCCCATA-3 (Forbi et al.2013 (link)). Primers were made in 25 μM concentrations and two microliter of each of the primers was added to a 50 μL reaction containing 10 μL of Red load Taq (Jena Bioscience, Jena, Germany), 4 μL of DNA and 32 μL of RNase free water. Thermal cycling was done in a Veriti Thermalcycler (Applied Biosystems, California, USA.) as follows; 94°C for 3 minutes followed by 45 cycles of 94°C for 30 seconds, 55°C for 60 seconds and 70°C for 40 seconds with ramp of 40% from 55°C to 70°C. This was then followed by 72°C for 7 minutes and held at 4°C till terminated. Reaction conditions were the same for both first and second round PCR except that DNA extract from the sample was used as template for first round PCR while first round PCR product was used as template for second round PCR. Finally, PCR products were resolved on 2% agarose gels stained with ethidium bromide and viewed using a UV transilluminator.

Pig feces from four distant (a separation of more than 3,500 km) industrialized feedlots that are representative large-scale swine farms located in (from south to north) Guangdong province, Sichuan province, Hebei province, and Heilongjiang province, respectively, in China. The fecal samples were obtained from representative large-scale swine farms with an animal intensity of 10,000 market hogs or more per year. A low-temperature box with ice was used to transport samples from the field to the laboratory; the samples were then immediately frozen and stored at −80°C for further analysis. Three to five fecal samples from each site were mixed, and a total of four mixed samples were prepared for each site. Fecal DNA was extracted using Qiagen DNA extraction kit (Qiagen, Germany) according to the manufacturer’s protocols. The DNA concentration and purity were quantified with TBS-380 and NanoDrop2000, respectively. DNA quality was examined with a 1% agarose gel electrophoresis system.

Genomic DNA was extracted from all K. pneumoniae strains (QIAGEN DNA extraction kit, QIAGEN, USA) and the rmpA and serotype-specific genes for the K1, and K2 capsular serotypes were amplified by polymerase chain reaction (PCR) as previously reported [16 (link),17 (link)].

SNP selection and genotyping has been previously described [22 (link)]. Briefly, PBMCs were isolated from blood samples of cases and controls by Ficoll-Paque gradient centrifugation following manufacturer’s protocol (Amersham Biosciences, Baie d’Urfé, Quebec, Canada). DNA was extracted from PBMCs by phenol-chloroform or Qiagen DNA extraction kit (Qiagen Inc., Hilden, Germany). The SNP rs1800734 was genotyped using a fluorogenic 5ʹ nuclease polymerase chain reaction (PCR) assay. It was also genotyped using Affymetrix GeneChip Human Mapping 100K and 500K platforms through the Assessment of Risk of Colorectal Tumours in Canada project [35 (link), 36 (link)]. Genotypes of the five OFCCR phase II samples used for bisulfite sequencing were confirmed by Sanger sequencing at The Centre for Applied Genomics (TCAG), The Hospital for Sick Children, Toronto, Canada. DNA from samples was amplified by PCR for the region in the MLH1 promoter encompassing rs1800734 using primer sequences: (forward) 5ʹ-CGCCACATACCGCTCGTAGTA-3ʹ and (reverse) 5ʹ-TCCGTACCAGTTCTCAATCATCTC-3ʹ. Sequencing was performed at TCAG using an internal primer (forward) 5ʹ-GTCATCCACATTCTGCGGGA-3ʹ.

Using the strategy reported previously (26 (link),27 (link)), we selected samples for further genetic assays on the basis of the relationship between the 2 pan-Plasmodium antigens (aldolase and lactate dehydrogenase [LDH]) and the HRP2/3 signal. Samples were selected if they completely lacked an assay signal for HRP2/3 or if the assay signal for HRP2/3 was atypically lower compared with the level of pAldolase or pLDH antigens.
We extracted total genomic DNA from 6-mm punches of selected DBS samples by using the QIAGEN DNA extraction kit (QIAGEN, https://www.qiagen.com) following the manufacturer’s instructions for blood dried on filter paper. The DNA was eluted in 150 μL of elution buffer, aliquoted, and stored at −20°C until further use.

DNA was extracted using the Qiagen DNA Extraction Kit (Qiagen; Hilden, Germany) according to the manufacturer’s instructions and quantified using high-sensitivity spectrophotometry (Nano Drop 1000, Labtech Ltd.; East Sussex, UK).
Multiplex single-tube PCR amplification was performed using a 20 μL reaction mixture of 4 components, including (i) distilled water (6 µL); (ii) genomic DNA samples (1 µL; 10 ng/μL), (iii) 2× KAPA2G Fast Multiplex Mix (Kapa Biosystems; Massachusetts, USA) (10 μL) and (iv) primer mixture (3 μL; 5 μM), c.1707_Fw_Wt, c.1707_Fw_Vr, c.1707_Rv, c.2168_Fw_Wt, c.2168_Fw_Vr and c.2168_Rv.
PCR was performed under the following condition: (i) initial denaturation step (95 °C; 3 min) and (ii) 28 cycles of the 3-step (95 °C; 15 s) → (60 °C; 30 s) → (72 °C; 30 s) sequence in the PCR Thermal Cycler (GeneAlas G02, Astec, Fukuoka, Japan).
Stability of this protocol was confirmed with PCR performed at a higher (50 ng/uL) and lower (5 ng/uL) DNA concentration at different annealing temperatures (59–60 °C or 61 °C).