Reagents were obtained and used as received. DNA templates and primers were obtained from Integrated DNA Technologies. The RNA oligonucleotide competitors and precursor RNAs, obtained from Dharmacon, were 2′-ACE-protected (where ACE is 2′-bis(acetoxyethoxy)-methyl ether) and were deprotected according to the manufacturer’s protocol. All RNAs were desalted using PD10 Sephadex (GE Healthcare) columns by first pre-equilibrating the column with 25 ml water, then loading the RNA and eluting with 10 ml water, collecting 1 ml fractions. Autoradiography images were obtained using a Typhoon 9410 variable mode imager (GE Healthcare) and quantified using Quantity One (Bio-Rad) software. All UV-vis measurements for RNA quantification were obtained using a Beckman Coulter DU800 UV-vis spectrophotometer by heating the RNA to 90 °C and measuring the absorption at 260 nm. Complementary DNA (cDNA) samples were quantified using an Agilent Technologies 2100 Bioanalyzer (Model: G1939A) and an Invitrogen Qubit 2.0 Fluorimeter. Subsequent sequencing was carried out on a Life Technologies Ion Proton sequencer with at least 200-fold coverage. EGM-2 Bullet Kit tissue culture medium obtained from Lonza (CC-3162) was used to grow HUVECs directly. All cells were cultured at 37°C in 5% CO2 in 100-mm-diameter dishes unless stated otherwise.
Pd10 sephadex
Manufactured by GE Healthcare
The PD10 Sephadex is a lab equipment used for desalting and buffer exchange of small molecules. It utilizes Sephadex, a dextran-based gel filtration medium, to separate molecules based on their size and molecular weight. The PD10 Sephadex is a pre-packed column that allows for convenient and efficient sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Rna oligonucleotide competitors and precursor rnas, by Horizon Discovery (2 mentions)
Bioanalyzer 2100, by Thermo Fisher Scientific (2 mentions)
Egm 2 bullet kit tissue culture medium, by Lonza (2 mentions)
Typhoon 9410, by GE Healthcare (2 mentions)
Qubit 2.0 fluorimeter, by Thermo Fisher Scientific (2 mentions)
Ion proton sequencer, by Thermo Fisher Scientific (2 mentions)
Quantity one, by Bio-Rad (2 mentions)
Du 800 uv vis spectrophotometer, by Beckman Coulter (2 mentions)
3 protocols using pd10 sephadex
1
RNA Oligonucleotide Purification and Quantification
2
Adenovirus-Mediated Prohibitin 2-DN Delivery
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The prohibitin 2 dominant-negative fragment (prohibitin 2-DN, aa201–299) was subcloned into an adenovirus vector pAdTrack-CMV. Subsequently, the recombinant adenovirus plasmids pDC316-mCMV-prohibitin 2-DN were transfected into HEK293A cells to yield the final expression clone Ad-prohibitin 2-DN. Ad-prohibitin 2-DN was amplified by infecting HEK293A cells and purified by PD-10 Sephadex (GE healthcare, Marlborough) precipitation. An adenovirus vector carrying green fluorescence protein (Ad-GFP) was applied as negative control. For in vivo studies, 1 × 1010 pfu of adenovirus dissolved in 30% pluronic gel solution were perivascularly delivered to the carotid arteries post balloon injury as described previously21 (link).
3
RNA Oligonucleotide Purification and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reagents were obtained and used as received. DNA templates and primers were obtained from Integrated DNA Technologies. The RNA oligonucleotide competitors and precursor RNAs, obtained from Dharmacon, were 2′-ACE-protected (where ACE is 2′-bis(acetoxyethoxy)-methyl ether) and were deprotected according to the manufacturer’s protocol. All RNAs were desalted using PD10 Sephadex (GE Healthcare) columns by first pre-equilibrating the column with 25 ml water, then loading the RNA and eluting with 10 ml water, collecting 1 ml fractions. Autoradiography images were obtained using a Typhoon 9410 variable mode imager (GE Healthcare) and quantified using Quantity One (Bio-Rad) software. All UV-vis measurements for RNA quantification were obtained using a Beckman Coulter DU800 UV-vis spectrophotometer by heating the RNA to 90 °C and measuring the absorption at 260 nm. Complementary DNA (cDNA) samples were quantified using an Agilent Technologies 2100 Bioanalyzer (Model: G1939A) and an Invitrogen Qubit 2.0 Fluorimeter. Subsequent sequencing was carried out on a Life Technologies Ion Proton sequencer with at least 200-fold coverage. EGM-2 Bullet Kit tissue culture medium obtained from Lonza (CC-3162) was used to grow HUVECs directly. All cells were cultured at 37°C in 5% CO2 in 100-mm-diameter dishes unless stated otherwise.
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