Nanoplexes were prepared as described previously (Elangovan et al., 2014 (link)). Briefly, two 500 μL solutions containing either 130 μg of 25 kDa branched PEI (Sigma-Aldrich) or 100 μg of pDNA were prepared in DNAse/RNAse-free water (ThermoFisher Scientific, Waltham, MA, USA). The PEI solution was added to the pDNA solution, vortexed for 30s, and incubated for 30 minutes to allow for complexation between the pDNA and PEI. The resulting 1 mL nanoplex solution had a nitrogen (N) to phosphate (P) ratio (N/P ratio) of 10, which was shown to yield maximum transfection efficiency with minimal cytotoxicity (D’Mello et al., 2016 (link); Elangovan et al., 2014 (link)). Varying volumes of 40% sucrose solution in DNAse/RNAse-free water (ThermoFisher Scientific) were added to the nanoplex solution to yield the desired sucrose concentrations (10%, 7.5%, 5%, 2.5%, 2%, 1%).
Dnase rnase free water
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom
DNase/RNase-free water is a high-purity water product designed for use in sensitive molecular biology applications. It is free of detectable DNase and RNase enzymes, which can degrade nucleic acids. This water is suitable for use in various laboratory procedures where RNase-free and DNase-free conditions are required.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (8 mentions)
Isopropanol, by Merck Group (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Poly a tailing kit, by Thermo Fisher Scientific (4 mentions)
Mini quick spin rna column, by Roche (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Trizol, by Merck Group (3 mentions)
Chloroform, by Merck Group (3 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (3 mentions)
Rnaiso plus, by Takara Bio (3 mentions)
Steponeplus system, by Thermo Fisher Scientific (3 mentions)
Lmd7000 laser microdissection system, by Leica (2 mentions)
Lightcycler 1, by Roche (2 mentions)
Thunderbird sybr qpcr mix, by Toyobo (2 mentions)
Spss version 16, by IBM (2 mentions)
Rotor gene real time pcr system, by Qiagen (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Mirneasy mini kit, by Thermo Fisher Scientific (2 mentions)
Qiaamp dna mini and blood mini handbook, by Qiagen (2 mentions)
111 protocols using dnase rnase free water
1
Preparation of PEI-based Nanoplexes
2
RNA Extraction and Reverse Transcription
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two to five hundred nanograms of total RNA was used in reverse transcription (RT) reactions. RNA was mixed with 0.5 µL of 100 mM oligo(dT)18 primer (Sigma), and completed to a final volume of 5 µL using RNase/DNase-free water (Ambion). This mixture was heated up for 5 min at 70 °C, and then chilled to 4 °C. During the cooling step, 15 µL of the RT reaction solution is added to the mixture. The added RT reaction solution contains 4 μL of 5X First Strand Buffer (250 mM Tris–HCl pH 8.3, 375 mM KCl and 15 mM MgCl2), 2 μL of 0.1 M Dithiothreitol (DTT), 1 μL of 10 mM dNTPs, 0.1 μL Superscript III reverse transcriptase (Invitrogen) and 7.9 μL RNase/DNase-free water (Ambion). The reaction was continued by an incubation step at 25 °C for 5 min, followed with 90 min incubation at 42 °C and 15 min incubation at 70 °C before finally holding the reaction at 4 °C.
3
Laser Microdissection of Vascular Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A Leica LMD 7000 Laser Microdissection System (Leica Microsystems) was used to isolate cells from the tissue sections. The tissues were visualized under a microscope, target cells were selected and encircled on the computer monitor using a mouse, and then the computer program guided a UV laser (337-nm wavelength) to cut the slide foil with the target cells (Figure 1 ). The analysis of matched tissue from the same rat avoids confounding effects of the genetic background, previously described as methylation quantitative trait loci [24 (link)]. The targeted cells fell into the 0.5-ml thin-wall DNase-free PCR tube caps (BIO plastics) filled with 12 µl DNase/RNase free water (Invitrogen, 75-0024) located beneath the visualized tissue section. The dissection conditions were optimized to obtain a clean, narrow excision of the selected cells: 40-XT objective at power 35 to 45 and speed 3 to 4. Collected samples were centrifuged at full speed (>10,000 ×g) for 5 minutes, and stored at –20°C. In the injured arteries, we used the laser to specifically isolate VSMC-like layer in the neointima leaving the endothelial layer intact; we also isolated the resident VSMC population in the medial layer as the control.
4
Comparative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from cells using the RNeasy mini kit (QIAGEN AB, Sollentuna, Sweden) and cDNA synthesis was performed using the ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan).
For qualitative PCR, the cDNA was amplified using PCR with specific primers. The following primers were used:
For qRT-PCR, each reaction mixture was diluted five-fold with DNase/RNase-free water (Invitrogen, Carlsbad, CA, USA), and 4 μL of each mixture was subjected to PCR. The reactions were run using THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) on a Light Cycler 1.5 (Roche, Indianapolis, IN, USA). The comparative Ct (ΔΔCt) method was used to determine fold-changes in expression using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference. Each sample was run in triplicate. The cycling conditions were as follows: initial denaturation at 98 °C for 5 min, followed by 45 cycles at 98 °C for 15 s, 60 °C for 30 s and 72 °C for 60 s. The experiments were performed in triplicate.
For qualitative PCR, the cDNA was amplified using PCR with specific primers. The following primers were used:
For qRT-PCR, each reaction mixture was diluted five-fold with DNase/RNase-free water (Invitrogen, Carlsbad, CA, USA), and 4 μL of each mixture was subjected to PCR. The reactions were run using THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) on a Light Cycler 1.5 (Roche, Indianapolis, IN, USA). The comparative Ct (ΔΔCt) method was used to determine fold-changes in expression using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference. Each sample was run in triplicate. The cycling conditions were as follows: initial denaturation at 98 °C for 5 min, followed by 45 cycles at 98 °C for 15 s, 60 °C for 30 s and 72 °C for 60 s. The experiments were performed in triplicate.
5
Genotyping of IL28B and TLR4 SNPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SNPs on IL28B (rs12979860) and TLR4 (rs4986791) were identified using a real-time PCR protocol based on the pre-validated TaqMan MGB™ probe for allelic discrimination assay (Applied Biosystems). Briefly, 1.25 μL of a 40X combined primer and probe mix (ABI/Life Technologies, USA) was added to 12.5 μL of 2X TaqMan® Universal PCR master mix (ABI/Life technologies, USA) in a 25 μL final volume of DNAse/RNAse-free water (Invitrogen/Life Technologies, USA) and template. The cycle conditions were: 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min. The last two steps were repeated 40 times. The PCR run was performed on Rotor Gene real-time PCR system (Qiagen, Santa Clarita, CA). Allelic discrimination plots were produced in Statistical Package for The Social Sciences (SPSS version 16.0; SPSS, Chicago, IL).
6
Cell Proliferation Quantification in ES Spheroids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess cell proliferation, we monitored DNA synthesis throughout the culture. ES spheroids (cultured alone or within alginate microcapsules) were sampled from shake flasks at specific time points. ES spheroids were recovered from capsules by using a chelating solution (10 mM HEPES, 100 mM EDTA, pH 7.4) and recovered by centrifugation at 50× g for 1 min. Pellets were resuspended in 1 mL of DNAse/RNAse-free water (Invitrogen) and stored at −80 °C until analysis. Once all samples were collected, they were subjected to 30 min of ultrasounds to lyse cells and release DNA. Cell proliferation was measured by the amount of dsDNA present in the samples using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen), following the manufacturer’s instructions. dsDNA quantification was normalized by the PrestoBlueTM assay performed in capsules before the recovery of the spheroids. Data are presented as the fold change of the dsDNA content compared to day 0, set as 1. The non-parametric Kruskal Wallis test was performed for statistical analysis.
7
Isolation and Quantification of Plasma cfDNA and Germline/Tumor gDNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell-free DNA (cfDNA) was isolated from 5 mL plasma using the QIAamp Circulating Nucleic Acid kit (Qiagen), as per the manufacturer’s protocol. Leucocyte buffy coats collected after the 1500g K2-EDTA vacutainer centrifugation were used to prepare germline genomic DNA (gDNA) using the QIAamp DNA Blood Mini Kit (Qiagen) as per the manufacturer’s instructions. Fresh frozen tumour tissue samples were available from four patients, from which tumour gDNA was isolated using a NucleoSpin Tissue Kit (Machery Nagel). Both gDNA and cfDNA were eluted in ultrapure DNase/RNase-free water (Invitrogen). DNA concentration was determined using a Qubit Fluorometer (dsDNA HS or dsDNA BR Assay Kits, ThermoFisher Scientific, MA, USA).
8
Lipid-Peptide Nanoparticle Formulations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lipids (Supplementary Fig. 1 ) 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-sn-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine (DOPE) and Rhodamine-DOPE were purchased from Avanti Polar Lipids (Birmingham, Alabama, USA). The peptide sequences K16GACSERSMNFCG (K16E) and K16GACYGLPHKFCG (K16Y) were purchased from China Peptides (Shanghai, China) and dissolved to 10 mg/mL in DNase/RNase free water (Invitrogen, Paisley, UK). The plasmid pCI-Luc comprised the luciferase gene of pGL3 (Invitrogen, Paisley, UK) subcloned into the eukaryotic expression vector pCI (Promega, Southampton, UK).
9
Laser Microdissection of Ovarian Epithelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The microdissection of ovarian epithelial cells from normal human ovarian tissue was performed using a laser-directed and computer-assisted microdissection microscope (Leica AS LMD 7000; Leica Microsystems) (Figure 7, A–C ) with a pulsed 337 nm UV laser. Captured single cells were dissected out and collected into individual tubes (EU single thin-wall 0.2 ml tube with cap, BIO plastics) filled with 20 µl DNase/RNase-free water (Invitrogen, 75-0024). The tubes were centrifuged at full speed (>10,000×g) for 5 minutes, after which the top 11 µl of water was discarded. All experiments were carried out in duplicate.
10
IFNL3 Polymorphism Detection via Real-Time PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The IFNL3 polymorphism (rs12979860 C > T) was detected using an allelic discrimination real-time-PCR protocol based on the pre-validated TaqMan-MGB™ probe (Applied Biosystems, Foster City, California, US). Briefly, 1.25 μL of a 40X-combined-primer and probe-mix (ABI/Life Technologies, USA) was incorporated to 12.5 μL of 2X TaqMan® Universal-PCR-master-mix (ABI/Life-Technologies, USA) and DNA template and then complete to 25 μL final volume with DNase/RNase-free-water (Invitrogen/Life-Technologies, USA). The thermal profile of amplification was 95 °C for 10 min, 95 °C for 15s, and 60 °C for 1 min. The final two steps were performed 40 times. The Rotor-Gene real-time PCR machine (Qiagen, Santa Clarita, CA) was used for the PCR run. The Statistical Package for The Social Sciences (SPSS version 16.0; SPSS, Chicago, IL) was used to create Allelic discrimination plots.
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!