Genomic DNA was extracted from all samples using the DNeasy Tissue kit (Qiagen, Valencia, CA, USA), according to protocols recommended by the manufacturer. Total RNA was extracted from the HCC cell lines and frozen tissue samples, which were pulverized in liquid nitrogen using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). To reduce the risk of genomic DNA contamination, 1 to 2 μg RNA was incubated with 2 U DNase I (Invitrogen, Carlsbad, CA, USA), 1 μL DNase buffer and, 0.4 μL RNase Out for 15 min at room temperature. The RNA concentration was determined by spectrophotometry, and the total RNA integrity was examined by visualization of the 28S and 18S ribosomal RNAs in a 1.2% agarose gel. First-strand cDNA was synthesized using the PrimeScript RT Reagent Kit (TaKaRa, Otsu, Japan) according to the manufacturer’s instructions.
Dnase buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
DNase buffer is a solution used in molecular biology applications to inactivate deoxyribonuclease (DNase) enzymes. DNase enzymes can degrade DNA, which is often undesirable in certain experimental procedures. The DNase buffer helps to inhibit the activity of these enzymes, ensuring the integrity of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Turbo dnase, by Thermo Fisher Scientific (10 mentions)
Rnaseout, by Thermo Fisher Scientific (4 mentions)
Rnase one, by Promega (4 mentions)
Dnase, by Thermo Fisher Scientific (4 mentions)
Rnase 1, by Thermo Fisher Scientific (3 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Baseline zero, by Illumina (2 mentions)
0.45 μm filter, by Merck Group (2 mentions)
Dntps, by Takara Bio (2 mentions)
Rnase free h2o, by Takara Bio (2 mentions)
First strand buffer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Na2edta, by Thermo Fisher Scientific (2 mentions)
Purelink viral rna dna mini kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dneasy tissue kit, by Qiagen (1 mentions)
Cfx96 real time pcr thermocycler, by Bio-Rad (1 mentions)
Am9260g, by Thermo Fisher Scientific (1 mentions)
20 protocols using dnase buffer
1
DNA and RNA Extraction for HCC Analysis
2
Single-Cell Viral Transcript Analysis
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At 3 days post-infection, cells were stained for sorting as described above. Single cells meeting lineage sort criteria were sorted into each well of a 0.2ml 96-well PCR plate containing 4μl of 0.5x PBS, 10mM DTT (Pierce no-weigh A39255), 1.2U RNase inhibitor (Lucigen 30281–2). After sorting, plates were sealed, centrifuged briefly to collect all material in the bottom of the well and stored at -80°C prior to analysis. Plates were thawed on ice and 2μl of DNase buffer (Invitrogen 18068–015) containing 0.5μl 10x buffer, 0.1U DNase, 0.4μl H2O) were added to each well. After incubation at room temperature for 15 minutes, EDTA (Thermofisher AM9260G) was added to a final concentration of 2mM and DNase was inactivated by incubation for 10 minutes at 65°C. One-Step RT-PCR reactions and no RT (NRT) controls were assembled using outer primers to GAPDH, LANA, and K8.1 as described above. 2μl of pre-amplified cDNA was used in the real time PCR reactions for GAPDH, K8.1, LANA as described above with the exception that the assay was multiplexed with all three targets using the same K8.1 probe sequence labeled with 5’Cy5 and 3’BHQ-2 quencher and analyzed on a Biorad CFX96 real time PCR thermocycler.
3
AAV2 Pretreatment for rCRISPR
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The affinity-purified AAV2 (Millipore Sigma) underwent pretreatment through capsid lysis at 95 °C for 15 min. Prior to performing rCRISPR, AAV dilutions were prepared in 1× DNase buffer (Invitrogen) supplemented with 0.001% Pluronic F-68.
4
Total RNA Extraction from Cork Oak Roots
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Total RNA was extracted from 50 mg of micropropagated clonal cork oak roots with the RNeasy kit from Qiagen, according to the instructions supplied by the manufacturer (Dudareva et al. 1996 (link)). Traces of DNA were removed with 2 μl DNase I (1 U/μL, Invitrogen), in the presence of 2 μl RNaseout (40 U/μL, Invitrogen) in 10 μl DNase buffer (200 mM Tris–HCl, pH 8.4, 20 mM MgCl2, 500 mM KCl, Invitrogen). RNA purity and integrity is essential for synthesis of full-length cDNA. Concentration of total RNA were determined by measuring the absorbance at 260 nm and the ratio of the absorbance at 260/280 nm was used to assess the RNA purity in a spectrophotometer MBA 2000 (Perkin Elmer). RNA was considered pure when a ratio of ~2.0 was obtained. As a routine procedure the integrity of total RNA was checked by electrophoresis in a denaturing 1.2% agarose gel, stained with ethidium bromide (Sambrook and Russell 2001 ).
5
Viral Nucleic Acid Extraction Protocol
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To reduce nonviral nucleic acids, 200 μl of filtered supernatant was treated with a nuclease mixture of 7 μl TURBO DNase (Ambion, Life Technologies, Grand Island, NY, USA), 3 μl Baseline‐ZERO DNase (Epicentre, Chicago, IL, USA), and 1 μl of diluted RNase T1 (Fermentas Canada Inc., Burlington, ON) in 7 μl 1× DNase buffer (Ambion). This mixture was incubated at 37°C for 90 min (Victoria et al., 2009 ; Zhang et al., 2014 ). DNase and Baseline‐Zero were inactivated by incubating for 20 min at 70°C. RNase T1 was inactivated during the first step of nucleic acid extraction. Viral nucleic acids were extracted from 200 μl of the DNase‐ and RNase‐treated product (Invitrogen Viral RNA/DNA Extraction kit; ThermoFisher Scientific, Mississauga, ON, Canada). In the purification procedure, 20 μl of RNase‐free water was used to elute nucleic acids.
6
Viral Nucleic Acid Extraction and Purification
7
Viral Nucleic Acid Purification Protocol
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The viral multiplex reagent (200 μl) was centrifuged at 12.000 ×
g for 5 min at 8 °C and the supernatant filtered through a 0.45 μM filter (Millipore, Billerica, MA, USA) to remove possible host cellular debris and bacteria. The filtrate was treated with a nuclease mixture of 14U turbo DNase (Ambion, Life Technologies, Grand Island, NY, USA), 3U Baseline-ZERO (Epicentre, Chicago, IL, USA) and 20U RNase One (Promega, Madison, WI, USA) in 1× DNase buffer (Ambion, Life Technologies, Grand Island, NY, USA) at 37 °C for 1.5 h to reduce background nucleic acids from the host cells and bacteria. Viral nucleic acids protected from digestion by viral capsids, were then extracted from ∼200 μl resulting solutions by different methods (Victoria et al., 2008 (link), Victoria et al., 2009 (link)).
g for 5 min at 8 °C and the supernatant filtered through a 0.45 μM filter (Millipore, Billerica, MA, USA) to remove possible host cellular debris and bacteria. The filtrate was treated with a nuclease mixture of 14U turbo DNase (Ambion, Life Technologies, Grand Island, NY, USA), 3U Baseline-ZERO (Epicentre, Chicago, IL, USA) and 20U RNase One (Promega, Madison, WI, USA) in 1× DNase buffer (Ambion, Life Technologies, Grand Island, NY, USA) at 37 °C for 1.5 h to reduce background nucleic acids from the host cells and bacteria. Viral nucleic acids protected from digestion by viral capsids, were then extracted from ∼200 μl resulting solutions by different methods (Victoria et al., 2008 (link), Victoria et al., 2009 (link)).
8
Identifying Nucleic Acid in MotB Samples
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MotB samples used to identify copurifying nucleic acid were purified using MotB purification Method I with the following modifications. Elution fractions from the chitin column that contained MotB as determined by SDS-PAGE were directly dialyzed into MotB Storage Buffer and stored at −80 °C. Protein was removed by chloroform:isoamyl alcohol (24:1) extraction, and sodium acetate and glycogen (Thermo Scientific, Waltham, MA, USA) were added to the aqueous phase at a final concentration of 496 mM and 0.17 mg/mL, respectively. The nucleic acid was then ethanol precipitated, resuspended in nuclease-free water, and quantified by NanoDrop 2000c (Thermo Scientific).
To identify nucleic acid, 1× DNase buffer (Ambion, Carlsbad, CA, USA) was added to ~300–400 ng nucleic acid followed by the addition of either 2 units of RNase-free DNase I (Ambion) or 100 µg of DNase-free RNase A and incubation at 37 °C for 10 min. Treated samples and the untreated control were electrophoresed on 0.8% (w/v) agarose gels at 120 V for 45 min in 1× TAE (Quality Biological) at room temperature. Gels were stained with ethidium bromide for 1 min and visualized using UV illumination. Two independent replicates were performed.
To identify nucleic acid, 1× DNase buffer (Ambion, Carlsbad, CA, USA) was added to ~300–400 ng nucleic acid followed by the addition of either 2 units of RNase-free DNase I (Ambion) or 100 µg of DNase-free RNase A and incubation at 37 °C for 10 min. Treated samples and the untreated control were electrophoresed on 0.8% (w/v) agarose gels at 120 V for 45 min in 1× TAE (Quality Biological) at room temperature. Gels were stained with ethidium bromide for 1 min and visualized using UV illumination. Two independent replicates were performed.
9
Detecting GalNAc in Bacterial LOS
10
Viral Nucleic Acid Extraction and RT-PCR Protocol
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Clearly, 14 U of Turbo DNase (Ambion, Austin, TX, USA), 25 U of Benzonase Nuclease (Novagen, San Diego, CA, USA), 20 U of RNase I (Fermentas, Ontario, Canada), and 10 × DNase buffer (Ambion) were added to 127 μL of the supernatants to a final volume of 150 μL, followed by digestion at 37°C for 1 h. After removing free nucleic acid and eliminating the contaminating host genomic DNA, the viral nucleic acid in the obtained products was extracted using a Virus Nucleic Acid Kit (Bioer Technology, Hangzhou, China) according to the manufacturer's instructions. The total viral nucleic acids were reverse transcribed using anchored random primers and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA). Anchored random primers (Table 2 ) were added separately to the viral nucleic acid and incubated at 75°C for 5 min and placed on ice for 5 min for denaturation. To acquire the reverse transcribed product, 40 U of RNase OUT, 200 U of Superscript III reverse transcriptase, 1 μL of 0.1 M dithiothreitol (DTT), 1 μL of 10 mM dNTPs, 4 μL of 5 × first strand buffer, and RNase-free H2O were added to a final volume of 20 μL. It was incubated at 25°C for 10 min followed by 50°C for 60 min, and 75°C for 10 min.
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