Genomic dna purification kit

Genomic DNA was extracted from the peripheral blood of subjects using a Genomic DNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.
The SNPs of MMP-2 C1306T were genotyped by polymerase chain reaction (PCR). The following primers were used: MMP-2 forward, 5'-CTTCCTAGGCTGGTCCTTACTGA-3'; MMP-2 reverse, 5'-CTGAGACCTGAAGAGCTAAAGAGCT-3'; TIMP-2 forward, 5'-CGTCTCTTGTTGGCTGGTCA-3'; TIMP-2 reverse, 5'-CCTTCAGCTCGACTCTGGAG-3'. PCR products were then separated by 1.5% agarose gel electrophoresis. β-Actin is used as internal control. The purified PCR products were sequenced using an ABI377 automatic sequencer to identify the variants. To ensure the quality of the sequencing, 10% of the patient group and the control group, respectively, were randomly selected and genotyped twice. The reproducibility was 100%.

N. winogradskyi DNA was prepared from cells grown in mixotrophic nitrobacter medium at 26 °C using a Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, USA), according to the manufacturer’s instructions. The DNA concentrations were determined using a Nanodrop spectrophotometer (Nanodrop Technologies, Rockland, DE). DNA isolated from N. winogradskyi was used as the template to amplify the nwiI gene using the following primers: 5′- CAGGATCCATGATTCACATCGTAACGGC-3′ (nwiI-forward) and 5′- CCGCTCGAGCTAGGCACGAAGCCGC-3′ (nwiI-reverse). The nwiI-forward primer included a BamHI restriction site, and the nwiI-reverse primer included an XhoI restriction site (underlined). The PCR conditions were 2 min at 95 °C followed by 30 cycles of 30 s at 95 °C, 30 s at 50 °C, and 1 min at 72 °C with a final step of 10 min at 72 °C. The amplified nwiI gene was cloned into pGEX-4T-1 (GE), according to the manufacturer’s instructions. The recombinant pGEX-4T-1 plasmid containing nwiI was termed pGEX-nwiI. pGEX-nwiI was transformed into E. coli BL21(DE3), and the transformant was grown on LB medium containing ampicillin (100 μg/ml) at 37 °C. The detailed characteristics of the bacterial strains and plasmids used in this study are presented in Supplementary Table S2.

Total genomic DNA was extracted from green leaves and seedlings using the “Genomic DNA Purification Kit” (Thermo Fisher Scientific, Vilnius, Lithuania) according to the manufacturer’s protocol. The DNA concentration and purification degree were determined using the Implen Nano Photometer NP60 spectrophotometer (Implen, Munich, Germany). Fifteen individual plants of each mutant line were used to estimate the genetic heterogeneity.