Wizard sv gel pcr clean up system

Four SYBR master-mix kits were evaluated: ssoAdvanced™ Universal SYBR® Green Master-Mix (Bio-Rad), QuantiNova SYBR® Green PCR Kit (QIAGEN), PowerUp SYBR® Green Master-Mix (Applied Biosystems) and RT² SYBR® Green qPCR Master-Mix (QIAGEN). RPL13a, SDHA, TBP, and IFN-γ primer (Table 2) amplicons were purified by Wizard SV Gel & PCR CleanUp System (Promega) and quantified by NanoDrop 2000 Spectrophotometer (ThermoFisher). Master-mix reaction efficiency was calculated by amplification of amplicons titrated from 10⁷ to 10¹ copies/reaction, with primers at 250, 500, or 750 nM (45 (link)).

Microalgal biomass was collected at the late exponential phase of cultivation for DNA extraction. DNA extraction was performed using a phenol:chloroform method. After extraction, genomic DNA within the 18S rRNA region was amplified on a PCR machine by using the following primers: Forward 5′-GCGGTAATTCCAGCTCCAATAGC-3′ and Reverse 5′-GACCATACTCCCCCCGGAACC-3′. The process followed was developed by Lim et al. (2012 (link)). PCR templates were then purified by using a Wizard SV Gel PCR Clean-Up System (Promega). For sequencing preparation, 5 μL of a 25 ng μL⁻¹ PCR product were combined with 1 μL of a 10 μM solution of each of the above primers. The reaction was topped up to 12 μL with Millipore water in a 1.5 mL tube and sent to the Australian Genome Research Facility (AGRF) at The University of Queensland for sequencing and analysis. The DNA sequence data was compared with Genbank entries for classification.

Chromatin immunoprecipitation (ChIP) experiments were performed essentially as described previously (30 ). In brief, ∼2.5 × 10⁷ ES-2 cells were treated with formaldehyde, quenched and harvested in lysis buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.9; 7.2 mM KOH; 150 mM KCl; 5 mM MgCl₂; 0.5% NP-40; protease inhibitors). Nuclei were enriched by centrifugation and lysed in ChIP-buffer (50 mM Tris-Cl, pH 8.0; 10 mM ethylenediaminetetraacetic acid (EDTA); 1% sodium dodecyl sulphate (SDS); protease inhibitors) before chromatin was sheared by sonification. For ChIP, 25 μg of sheared chromatin was incubated with control (anti-IgG, Abcam ab171870) or anti-SRF (NEB 5147) antibodies overnight in dilution buffer (10 mM Tris-Cl, pH 8.0; 150 mM NaCl; 1 mM EDTA; 0.1% SDS; 1% Triton X-100; protease inhibitors). Upon extensive washing, chromatin was eluted in elution buffer (50 mM Tris-Cl, pH 8.0; 10 mM EDTA; 1% SDS), treated with Proteinase K and cross-linking was reversed overnight. DNA was finally eluted using the WIZARD^®SV Gel & PCR Clean-Up System (Promega A9281) according to the manufacturer’s protocol and analyzed by quantitative real-time PCR (qPCR). RNA immunoprecipitation (RIP) and quantitative RT-PCR analyses were performed essentially as recently described (6 (link)). Primers are summarized in Supplementary Table ST5.

The pmScarlet_C1 plasmid (Plasmid #85042, Addgene) was used as a backbone. Total RNA was extracted from Hs578T cells using RNeasy plus mini kit (Qiagen) followed by cDNA synthesis using the RevertAid H minus first strand cDNA synthesis kit (Thermo Fisher Scientific) both according to the manufacturer’s protocol. To enrich for the CDCA5 intron retained product, the procedure was repeated 3 days after NHP2L1 knockdown. CDCA5 was amplified using the NEBQ5 hotstart master mix (New England Biolabs) using restriction site overhang primers (Forward: 5′-TAAGCAGAATTCATCTGGGAGGCGAACGC-3′, Reverse: 5′-TGCTTAGGATCCTCATTCAACCAGGAGATCAAACTGC-3′). PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega). CDCA5 inserts and pmScarlet_C1 backbone were enzymatically cleaved using EcoRI-HF and BamHI restriction enzymes (New England Biolabs), loaded on a agarose gel and purified with the Wizard SV Gel PCR Clean-Up System (Promega). Inserts and backbones were ligated using T7 DNA ligase (New England Biolabs) followed by transformation in C2987I electrocompetent bacteria (New England Biolabs). Colonies were screened for the desired product and validated by Sanger sequencing. Transfections were performed in MDA-MB-231 using Lipfectamine 3000 (Thermo Fisher) according to the manufacturer’s protocol.

Genomic DNA from soil and sediment samples were extracted from ∼ 0.33 g subsamples after freeze drying. The DNA from snow samples was extracted from 300–500 ml thawed snow water after being filtered through a 0.22 μm filter. DNA was extracted with a Fast DNA SPIN Kit for Soil (MP Biomedicals, United States), quantified and assessed for quality using a Nano Drop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, United States). PCR amplification of the anammox bacteria hydrazine synthase (HZS) was done using the hzsB_396F and hzsB_742R primer pairs (Wang et al., 2012 (link)). Amplification was performed in 50 μl reaction mixtures containing 5 μl 10 × buffer, 4 μl dNTP (2.5 mmol/L), 1 μl of each primer (10 mmol/L), 0.5 μl BSA, 0.25 μl Taq polymerase (2.5 U) (Takara, Japan) and 2 μl of the DNA template, and dd H₂O made up to 50 μl. The amplification thermal protocol consisted of following steps: 10 min at 95°C, then 35 cycles of 60 s at 95°C, followed by 60 s at 59°C, and 45 s at 72°C, and an extension of 10 min at 72°C. The PCR products were purified in 1% agarose gel after electrophoresis using the Wizard SV Gel & PCR clean-up system (Promega, United States) before multiplexing sequencing using the Hiseq 2500 platform (Illumina, United States).