Minelute 96 uf pcr purification kit

Genomic DNA from microdissected frozen tumor tissues and corresponding lymphocyte were analyzed in parallel. Briefly 250 ng DNA was digested with XbaI (New England Biolabs Inc., Ipswich, MA, USA), ligated to the adaptor, and amplified by polymerase chain reaction (PCR) using a single primer. After purification of PCR products with the MinElute 96 UF PCR purification kit (Qiagen, Valencia, CA, USA), amplicons were quantified, fragmented, labeled and subsequently hybridized on Affymetrix GeneChip1 Mapping 10 K 2.0 SNP microarrays following the manufacturer’s instructions (Affymetrix Inc., Santa Clara, CA, USA). After washing and staining, the arrays were scanned for data analysis.

Genomic DNA was extracted from the 395 pelleted samples (a single cecal lavage pellet was lost during handling) using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) with a 30-s beat-beating step in a Mini-Beadbeater-16 (BioSpec Products, Bartlesville, OK, USA) [53 (link)]. Polymerase chain reaction amplification of bacterial 16S rRNA genes was performed using PCR primers (F515/R806) targeting the V4 hypervariable region, with the reverse primers including a Golay barcode [54 (link)]. PCR products were purified using the MinElute 96 UF PCR Purification Kit (Qiagen, Valencia, CA, USA). DNA sequencing (100 bp reads) was performed using an Illumina HiSeq 2000 (Illumina, Inc., San Diego, CA, USA) as previously described [55 (link)]. Raw sequence data was demultiplexed and filtered in QIIME v1.9.1 using split_libraries_fastq with q=19 [56 (link)]. Deblur v1.1.0 was used with default parameters and min-reads 10 to denoise the data into amplicon sequence variants (ASVs) [57 ]. Taxonomy was assigned using the RDP classifier implemented in the assignTaxonomy function of the R package dada2 v1.16.0 and the Silva v138 database [58 (link), 59 (link)]. Three samples with fewer than 50,000 sequences were excluded from the analysis. The sequence depth of the remaining samples ranged from 50,862 to 821,276, with a mean depth of 411,244.

The DNA was sequenced using NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) as PCR primers for amplification of D1/D2 region of LSU rDNA [24 ]. PCR amplification was performed in 20 μL reaction, containing 10 μL of GoTaq® Green Master Mix (Promega), 10 pmol of each primer, and 2 μL of 1–20 ng/μL extracted DNA, on GeneAmp® PCR System 9700 (Applied Biosystems) or iCycler (BioLad). The PCR program was as follows; an initial denaturation at 94 °C for 5 min, followed by 36 cycles of 30 s at 94 °C, 30 s at 52 °C, 1 min at 72 °C, and a final extension of 5 min at 72 °C. The PCR-amplified fragment was visualized by electrophoresis on agarose and staining with ethidium bromide. Purification of the fragment was performed using MinElute® 96 UF PCR Purification Kit (Qiagen) according to the manufacturer's instructions. The purified fragment was resuspended in 50 μL of 10x diluted TE buffer. The nucleotide sequences of PCR-amplified fragment were determined by Sanger-sequencing using the ABI PRISM® 3130xl Genetic Analyzer (Applied Biosystems) following the manufacturer's instruction. Sequence data were corrected by manual inspection whenever needed, and aligned using BioEdit Sequence Alignment Editor version 7.1.3.0 [26 ].