Qiaamp tissue kit

High-molecular-weight genomic DNA from CRC tumor samples and corresponding normal tissue samples was purified using the QIAamp Tissue kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. The yield and purity were determined using a Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

DNA from gastric biopsies was extracted using the QIAamp® tissue kit (Qiagen, Germany) according to the manufacturer´s instructions. For detection of the H. pylori, PCR assays were performed using one set of oligonucleotides Hpx1/Hpx2 that amplifies a 150-bp fragment corresponding to 16S-rRNA from H. pylori (Table 1). In each experiment, positive (strain 26695) and negative (water) controls were included.

DNAs were extracted from fecal and milk samples using QIAamp tissue kit and QIAamp blood mini kit (Qiagen, Hombrechtikon, ZH, Switzerland), respectively, according to the manufacturer’s instructions with some modifications^{52 (link),53 (link)}.

DNA was isolated from infected cells in culture using the QIAamp Tissue Kit (Qiagen). The entire env gene was amplified by PCR using Taq DNA Polymerase (Promega, Madison, WI, USA) with the primers 5′GGGACGAGGTTATGCCGCTG-3′ (~50 bp upstream of Asp718 site) and 5′TACCACCACCCATGTACTGCC-3′ (just downstream of the env gene). Each Taq PCR contained 1.25 μL 10× PCR buffer (final concentration, 50 mm Tris-Cl, pH 8.3, 50 mM KCI, 7 mM MgCl₂ 1.1 mM β-mercaptoethanol), 1.25 μL of 1.7 mg/mL BSA, 0.5 μL of each dNTP at 25 mM, 0.5 μL of each primer (A₂₆₀ = 5), 6.0 μL H₂0, and 1.0 μL of DNA (genomic DNA ~100 ng/μL; plasmid DNA ~2 ng/μL). The reactions were heated to 90 °C for 1 min and initiated by the addition of 1.5 μL of Taq DNA Polymerase diluted 1:10 v/v (0.75 units). Thirty cycles of PCR were carried out as follows: 90 °C for 40 s, then 59 °C for 80 s. The amplified products were separated by agarose gel electrophoresis, the ~2.0 kb product purified and cloned into pCR2.1-TOPO using the TOPO TA Cloning kit (Invitrogen).
The nucleotide sequence of the env genes was determined by the Mayo Clinic Molecular Biology Core on a Perkin Elmer ABI PRISM 377 DNA sequencer (with XL upgrade) with PE Applied Biosystems ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit and AmpliTaq DNA Polymerase (PE Applied Biosystems, Foster City, CA, USA).

Cryopreserved tissue sections of 8–10-μm thickness, fixed in 70% ethanol and stained with a 0.05% w/v solution of toluidine blue, were microdissected using a laser micromanipulator (PALM MicroLaser Systems, ZEISS, Göttingen, Germany). Microdissected fragments were collected in PALM Adhesive Caps. Tumor DNA was isolated from these fragments using QIAamp Tissue Kit (QIAGEN, Hilden, Germany). In all cases, normal autologous DNA was obtained from peripheral blood lymphocytes (PBLs) using QIAGEN DNA isolation kit following the manufacturer’s protocol.