After protein analysis, the samples were concentrated to an approximate volume of 200 μL using heat and air. RNA precipitation was carried out by adding a 1:1 volume of phenol:chloroform solution (Sigma) to the samples, followed by vigorous shaking for 1 min. The samples settled at room temperature for 10 min before centrifuging at 13,000 rpm for 20 min. The aqueous phase was transferred to a new Eppendorf tube. A 1:10 volume of 3 M NaAc (Ambion) and a 2.5 volume of cold 100% EtOH (Sigma) was added to the samples. The samples were put in either −80°C for 20 min or −20°C for several hours. The samples were then spun at 13,000 rpm for 15 min at 4°C. The supernatant was discarded, and the RNA pellet was washed with ice-cold 70% EtOH. The samples were centrifuged at 8,000 rpm for 10 min at 4°C. The supernatant was discarded, and the RNA pellet dried. The pellet was resuspended in 50 μL of RNase-free water (Gibco). The samples were heated at 55°C for 5 min, and then RNase treated by adding 1 U of RNase A/C (Thermo Fisher) per manufacturer's instructions.
Phenol chloroform solution
Manufactured by Merck Group
Phenol:chloroform solution is a liquid reagent used in molecular biology and biochemistry laboratories. It is primarily used for the extraction and purification of nucleic acids, such as DNA and RNA, from biological samples. The solution consists of a mixture of phenol and chloroform, which effectively separates the aqueous and organic phases, allowing for the isolation of the desired nucleic acids.
Automatically generated - may contain errors
3 protocols using phenol chloroform solution
1
RNA Isolation and Purification Procedure
2
DNA Methylation Profiling in Silver-Russell Syndrome
3
Evaluating DNA Protection by Lipid Nanoparticles
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To assess the DNA protection ability of LNPs, we developed an assay based on DNase I treatment. Cationic or neutral LNPs containing 300 ng or 500 ng of pDNA or naked pDNA at the same amounts were incubated with 1 U/µL DNase I (MO303, New England Biolabs, Ipswich, Massachusetts) or water for 10 minutes at 37°C. The reaction was blocked by adding 5 mM EDTA and incubating for 10 minutes at 75 °C. Subsequently, DNA was extracted from the LNPs in 1% Triton-X 100 and purified by adding an equal volume of phenol/chloroform solution (P4557, Sigma-Aldrich). After emulsification, samples were centrifuged for 2 minutes at 16,200*g. The aqueous upper phase was transferred into another tube and DNA precipitated in 0.3 M Potassium acetate and 70% cold Ethanol. Samples were incubated overnight at −20°C, then centrifuged for 10 minutes at 16,200*g and 4°C. DNA pellets were washed with absolute Ethanol air dried and finally resuspended in water.
For quantification, all samples with purified pDNA were digested for 1 h at 37 °C with HindIII restriction enzyme (R0104, New England Biolab) and run on a 1% agarose gel. The internal standard curve was based on 3 concentrations of naked pDNA similarly digested and loaded into each gel. Bands were quantified using the Image Lab software (Bio-Rad).
For quantification, all samples with purified pDNA were digested for 1 h at 37 °C with HindIII restriction enzyme (R0104, New England Biolab) and run on a 1% agarose gel. The internal standard curve was based on 3 concentrations of naked pDNA similarly digested and loaded into each gel. Bands were quantified using the Image Lab software (Bio-Rad).
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