Phenol-chloroform extraction

Purify linearized plasmid by excision of the band from the gel (optional) and phenol/chloroform extraction. 

The in vitro transcribed RNAs are phenol-chloroform extracted, ethanol-precipitated, and washed once with 70% ethanol. The RNA pellet is resuspended in 30 1 H20 and loaded to DyeEx 2.0 spin columns (Qiagen) to remove free nucleotides the RNA is then quantified by spectrophotometry. Approximately 20 to 30 pg of RNA is obtained from one reaction. For the initial preparation of transcripts, we would examine the quality and quantity of synthesized RNA by separating them on 1% denaturing agarose gels with total cell RNA preparation as a size maker (28S and 18S ribosomal RNAs... 

After 2 h incubation of the prepared antibody beads with UV-crosslinked extract in a cold room, the beads are washed 4 x with 100 /A RIPA buffer (50 mMTris-HCl pH 7.5, 150 rnMNaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) and lx with genomic DNA lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 2.5 mM DTT, 0.5 mM spermidine, 1% Triton X-100). Approximately 300 /(I of PK solution (1 mg/ml proteinase K in genomic DNA lysis buffer and 0.2 U//A RNase inhibitor) is added to the total lysate previously kept on ice and the beads are then incubated at 37° for 30 min. Gently flick the tubes to resuspend the beads every 10 min during the incubation. After removal of the proteinase K solution, 300 /A of RNA extraction solution (4 M guanidine thiocyanate, 0.5% sarkosyl, and 25 mM sodium citrate, pH7) is added to the beads, incubated for 10 min and the supernatant is mixed with 30 fig yeast tRNA (as a carrier) and 30 fil of 3 M sodium acetate. The RNA solution is phenol-chloroform extracted, ethanol-precipitated, and the pellet washed once with 70% ethanol. The dry pellet is used for 1st strand cDNA synthesis, followed by PCR analysis. The removal of proteins... 

Anion exchange chromatography The reaction mixture is subjected to phenol/chloroform extraction to remove the T7 RNA polymerase using phenol equilibrated with 50 mM Na acetate (pH 4.5). After isopropanol precipitation, the pellets are resuspended in 20 mM MOPS buffer (pH 6.25) containing 350 mM NaCl. The excess unincorporated NTPs and the smaller abortive transcription products are removed by chromatography on anion exchange FPLC column (MonoQ 5/5 column, Amersham). 

Following linearization of plasmids (Fig. 13.5), the DNA is phenol chloroform extracted, passed through a Sephadex G50 spin column, and precipitated with 2.5 volume of ethanol and hjth volume of 3MNaOAc (pH 5.2). The pelleted DNA is washed with 70% ethanol, dried, and resuspended in water at a final concentration of 1 pg/pi and stored at —20°. 

Genomic DNA was isolated from each cell hne using standard techniques, which involved sequential washing in PBS and lysis in the presence of proteinase kj 0.4% SDS (Zuccotti and Monk, 1995). Following multiple phenol/chloroform extractions and ethanol precipitations, the amount from each line was... 

Isolate genomic DNA from tissne/cells nsing DNeasy tissue kit (Qiagen) or phenol chloroform extraction. 

Care should be taken to avoid several pitfalls. If the RNA has been produced by in vitro transcription, proteins should be removed by phenol/chloroform extraction. Otherwise the column bed may become coated with proteins and the column will lose its... 

Water-saturated phenol/chloroform is prepared in the following way. Phenol is fused by heating the botde in hot water (55-60°C). After addition of an excess amount ofwater, they are mixed well and left to allow separation of water from the mixture at room temperature. After water-phenol separation, the same amount of chloroform as the water-saturated phenol is added. These are then mixed well and left again to allow separation. Use the lower layer for phenol/ chloroform extraction. The upper water can be discarded if the volume is too excessive. 

Purify the amplified DNA fragment by phenol/chloroform extraction and ethanol precipitation. 

After incubation at 56°C for 1 h, phenol-chloroform extraction is performed by extracting the mixture 2-3 times with an equal volume of phenol see Note 11), an equal volume of phenohchloroform (1 1), and then twice with an equal volume of chloroform. 

Remove the reaction mixture from beneath the mineral oil, phenol/chloroform extract, chloroform extract, and precipitate the DNA by adding 25 pL 7 5M ammonium acetate and 150 pL ethanol. Precipitate at room temperature for 10 min. Centrifuge for 10 mm at 12,000 rpm in a microcentrifuge, wash the DNA pellet with 100 pL 70% ethanol, dry, and resuspend in 20 pL TE buffer Digest the DNA fragment with YhoI according to the supplier s instructions. 

The HC and LC PCR products are pooled separately, phenol/chloroform-extracted, and ethanol-precipitated. Following resuspension and, where appropriate, gel purification of PCR products, the DNA is quantitated, before restriction enzyme digestion and cloning into thepComb3 vector. Gel purification (see below for methods) may be necessary when there are a high number of background bands in the PCR reaction. This tends to be more of a problem... 

Chomczynski, P and Sacchi, N (1987) Single-step method of RNA isolation by acid guanidimum thiocyanate phenol chloroform extraction Anal Biochem 162, 156-159... 

Removal of proteins by phenol-chloroform extraction of cell lysate... 

The phenol used for RNA extraction should be of the highest purity (double distilled), as oxidation products of phenol can cause degradation of RNA. Before use, the phenol is saturated with DEPC-treated water, and the phases are allowed to separate. The pH of the top aqueous layer is tested with pH paper. If phenol is acidic, it is equilibrated with Tris buffer by mixing it with a volume of 1 M Tris-Cl, pH 7.0. After phase separation, the top buffer layer is removed and the phenol is equilibrated twice with water. Additives such as 8-hydroxyquinoline (0.1%) are used in phenol to inhibit the activity of nucleases. Caution should be exercised in the use of phenol, as it is corrosive and a suspected carcinogen. Use in a chemical hood is recommended. Phenol chloroform extraction should be carried out in con-... 

A recent development is to combine filtration with solid-phase extraction separation. These filter modules contain a unique silica gel membrane that binds up to 20 pg of DNA in the presence of a high concentration of chao-tropic salt and allow eventual elution in a small volume of low-salt buffer. They also contain an asymmetric laminar membrane with a gradation of pore sizes for efficient removal of material precipitated in the lysate. Such membrane filters eliminate time-consuming phenol-chloroform extraction and alcohol precipitation. The impregnation of silica in the membrane matrix also prevents the problems associated with loose resins and slurries. High-purity plasmid DNA eluted from such modules is ready to use and often needs no further precipitation, concentration, or desalting. 

Highly active in reversing inhibition by albomycin-ferrimycin type antibiotics. Phenol-chloroform extraction of 201 of Aspergillus melleus culture followed by further purification yields 4.65 g of substance. Also produced by A. terreus (63). 

Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162(1) 156—159 Liu CH, Ma WL, Shi R, Zhang B, Ou YQ, Zheng WL (2003) Gene expression study of saccaromyces cerevisiae with the Agilent 2100 Bioanalyser. Br J Biomed Sci 60(l) 22-25... 

A good stopping point in the end-labeling reaction is the ethanol precipitation step after the phenol/chloroform extraction. The RNA precipitation can be left at -20°C from an hour to overnight. 

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