For each sample, approximately 140 μL of viral transport medium was passed through a 0.22μm filter (Dot Scientific, Burton, MI, USA). Total nucleic acid was extracted using the Qiagen QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), substituting carrier RNA with linear polyacrylamide (Invitrogen, Carlsbad, CA, USA) and eluting in 30 μL of nuclease-free H2O.
Linear polyacrylamide
Manufactured by Thermo Fisher Scientific
Sourced in United States
Linear polyacrylamide is a high molecular weight polymer used as a sieving matrix in various electrophoresis techniques, such as DNA sequencing and protein separation. It provides a stable and consistent pore structure for the separation of biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp viral rna mini kit, by Qiagen (4 mentions)
Rna clean concentrator 5 kit, by Zymo Research (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Polyallomer centrifuge tube, by Beckman Coulter (1 mentions)
Dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Ar200 digital refractometer, by Reichert Technologies (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Rnase free water, by Qiagen (1 mentions)
Nanodrop nd 1000 spectrometer, by Avantor (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Am9937, by Thermo Fisher Scientific (1 mentions)
8 protocols using linear polyacrylamide
1
Viral Transport Medium Filtration and RNA Extraction
2
Sequence-Independent Viral RNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately 140 μL of VTM was passed through a 0.22μm filter (Dot Scientific, Burton, MI, USA). Total nucleic acid was extracted using the Qiagen QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), substituting carrier RNA with linear polyacrylamide (Invitrogen, Carlsbad, CA, USA) and eluting in 30 μL of nuclease free H2O. The sample was treated with TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 30 min and concentrated to 8μL using the RNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA). The full protocol for nucleic acid extraction and subsequent cDNA generation is available at https://www.protocols.io/view/sequence-independent-single-primer-amplification-o-bckxiuxn .
3
Viral RNA Extraction from Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For each sample, approximately 140 μL of viral transport medium was passed through a 0.22 μm filter (Dot Scientific, Burton, MI, USA). Total nucleic acid was extracted using the Qiagen QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), substituting carrier RNA with linear polyacrylamide (Invitrogen, Carlsbad, CA, USA) and eluting in 30 μL of nuclease-free H2O.
4
Isolation and Quantification of RNA from Brachiocephalic Arteries
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brachiocephalic arteries were lysed in 1 mL Trizole and homogenized before addition of 200 µL chloroform and incubation for 15 min. Samples were then centrifuged at 12000 g at 4 °C for 15 min to reach phase separation. The aqueous phase was retrieved and RNA was precipitated by addition of 2 µL of linear polyacrylamide (Invitrogen, Carlsbad, CA, USA) and 500 µL of isopropanol at −20 °C overnight followed by centrifugation at 12000 g at 4 °C for 15 min, before it was washed in 75% ethanol. RNA was dissolved in RNAse free water and concentration was measured by NanoDrop spectrophotometer. cDNA was synthesized from 600 ng of RNA using high capacity RNA to cDNA kit (Applied Biosystems, Stockholm, Sweden). Realtime quantitative PCR was performed using TaqMan Fast Advanced Master mix (Applied Biosystems, Stockholm, Sweden) with IL4 (Mm00445259_m1) or INFγ (Mm01168134_m1) gene expression assays from TaqMan (ThermoFisher Scientific). Results were calculated using ΔΔCT method. All results are shown as mean relative expression to control PBS-treated mice and controlled endogenously to the housekeeping genes β-actin (Mm00607939_s1) and GAPDH (Mm99999915_g1). IL-4 expression was below detection limit in the majority of samples.
5
SARS-CoV-2 Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The first confirmed case of SARS-CoV-2 in Dane county occurred on 30 January 2020. This early sample was processed using an early iteration of our SARS-CoV-2 sequencing protocol, as outlined here. All other samples included in this study were processed using a modified-version of the ARTIC-sequencing protocol, as outlined below. Approximately 140 µL of VTM was passed through a 0.22 µm filter (Dot Scientific, Burton, MI, USA). Total nucleic acid was extracted using the Qiagen QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), substituting carrier RNA with linear polyacrylamide (Invitrogen, Carlsbad, CA, USA) and eluting in 30 µL of nuclease free H2O. The sample was treated with TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 30 min and concentrated to 8 µL using the RNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA). The full protocol for nucleic acid extraction and subsequent cDNA generation is available at https://www.protocols.io/view/sequence-independent-single-primer-amplification-o-bckxiuxn .
6
Density Gradient DNA Fractionation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Density-gradient formation, fractionation, and clean-up was performed similarly to Morando and Capone (2018) . Briefly, DNA extracts were mixed with a gradient buffer (0.1 M Tris-HCl, 0.1 M KCl, 1 mM EDTA) and cesium chloride (7.163 M) in 3.3 ml polyallomer centrifuge tubes (Beckman Coulter) to a final density of 1.700 g ml -1 . Tubes were loaded into a TLN-100 near-vertical rotor (Beckman Coulter) and spun for 72 hours at 136,000 × g av and 20°C. Tubes were then fractionated in ~100 µl increments via displacement with mineral oil. Fraction densities were calculated using an AR200 Digital Refractometer (Reichert Technologies). DNA was precipitated from each fraction with PEG-NaCl (30% PEG, 1.6 M NaCl) and linear polyacrylamide (Invitrogen, Thermo Fisher Scientific), washed with 70% ethanol, dried, and resuspended in TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0). Recovered DNA was quantified using a Qubit fluorometer with a dsDNA BR Assay Kit (Invitrogen, Thermo Fisher Scientific).
7
Extracting High-Quality RNA from Dinospore Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRI Reagent–fixed and frozen dinospore cells were lysed using a Bio 101 FastPrep instrument (Thermo Savant, Illkirch, France) at maximum speed (6.5 m/s) for 2 × 45 s. Lysed cells were cooled on ice, and 200 μl of chloroform was added and vortexed for 20 s. The samples were transferred to a phase lock tube after 5 min of incubation at room temperature (Eppendorf, Hamburg, Germany) and incubated for another 5 min followed by centrifugation for 15 min at 13,000g and 4°C. The upper aqueous phase was transferred to a new tube and mixed with the same volume isopropanol, 1/10 volume of 3 M Na-acetate (pH 5.5; Ambion by Life Technologies, Carlsbad, CA, USA), and 2 μl of linear polyacrylamide (Ambion). Total RNA was precipitated for 90 min at −20°C and collected by centrifugation for 20 min at 13,000g and 4°C. The obtained pellet was washed twice, first with 1 ml of 70% ethanol (EtOH) followed by 1 ml of absolute EtOH; the RNA pellet was dried for 1 min at 37°C and resolved in 30 μl of RNase-free water (Qiagen, Hilden). RNA quality check was performed using a NanoDrop ND-1000 spectrometer (PEQLAB, Erlangen, Germany) for purity and RNA Nano Chip Assay on 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to examine the integrity of the extracted RNA.
8
RNA Isolation using TRIzol and Ethanol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated using a Trizol reagent. Bromochloro (0.1 M) was added per 1 ml TRIzol® Reagent (Ambion, 15596018). The tubes were vortex then centrifuged for 15 min at 12000 g at 4 °C. The aqueous phase was mixed with 2 μl of 5 μg/μl linear polyacrylamide (ambion) and 500 μl of isopropanol. The solution was then vortexed then left to incubate at room temperature. The solution was then centrifuged for 1 h at 20,000 × g at 4 °C. The supernatant was discarded followed by the pellet being washed with 1 ml of ice cold 200-proof ethanol. The sample was then vortexed followed by centrifugation for 30 min at 12,000 × g at 4 °C and the supernatant was discarded. The RNA pellet was left to air dry, then resuspended in 50 μl of RNAse free H2O (Ambion™ AM9937) and stored at ‒80 °C.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!