Phenol-chloroform

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... 

Discard the supernatant and resuspend the pellet in 650 1 RNase-free water. Add an equal volume of water-saturated phenol chloroform (5 1), pH 5.2. Vortex vigorously and spin at top speed for 5 min at room temperature. Take 500 pi of the aqueous phase into a new microtube tube. 

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°. 

Add 500pL of phenol chloroform isopropanol alcohol (25 24 1), mixed by vortex. 

In other Legionella species, such as L. jordanis, L. meceacherinii, and L. micdadei, such a-hydroxylated long-chain fatty acids as 2-hydroxy-27-oxo-octacosanoic acid [28 0(2-OH,27-oxo)], 2-hydroxy-29-oxo-triacon-tanoic acid [30 0(2-OH,29-oxo)], 2-hydroxyheptacosane-l,27-dioic acid [27 0(2-OH)dioic], and 2-hydroxynonacosan-l,29-dioic acid [29 0(2-OH)dioic] have been analyzed (161) in the phenol - chloroform - petroleum ether (PCP) extracts, indicating that they are constituents of lipopolysac-charide. 

Tris buffer Proteinase K Phenol-chloroform Ribonuclease Absolute ethanol... 

Remove the protein by extraction into phenol/chloroform. 

In this method the cells are lysed by incubation in a hypotonic solution, which leaves the nuclei intact. The cell debris and nuclei are pelleted by centrifugation leaving the cytoplasmic RNA free from DNA in the supernatant. The RNA is released from the polysomes by incubation with proteinase K and the protein extracted into phenol/chloroform. The RNA is then precipitated from the aqueous phase using ethanol. 

Extract the RNA transcript from solution using 25 24 1 (v/v) phenol/chloroform/isoamyl alcohol (conduct in a fumehood). First, add DEPC-treated water to bring the final volume to 400 pL subsequently, add 400 pL of phenol/chloroform/isoamyl alcohol to the reaction mix. Briefly vortex the tube and microfuge at 13,000 rpm ( 15,000 g) for 2 min at room temperature to separate the... 

Following DNase 1 digestion, the synthesized RNA is extracted with 1 volume of TE-saturated phenol/ chloroform, vortexed for 1 min and centrifuged in a microcentrifuge (12,000 x g) for 5 min. The aqueous phase is transferred to a fresh tube, 1 volume of chloroformusoamyl alcohol (24 1) is added, and the tube is vortexed for 1 min and centrifuged (12,000 x g) for 5 min. The aqueous phase is transferred into a fresh tube and cRNA precipitated by adding an equal volume of 5 M ammonium acetate and 2 volumes of ice-cold ethanol or an equal volume of isopropanol. The precipitation is carried on dry ice for a minimum of... 

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... 

Proteinase K, phenol chloroform, isopropanol, sodium acetate, glycogen, and ethyl alcohol. 

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

Recover the DNA by phenol chloroform exttaction followed by isopropanol/ sodium acetate plus glycogen precipitation. 

An equal volume of phenol/chloroform/isoamyl alcohol is added the mixture is vortexed for 1 min and stored on ice for 15 min (5 min for the isolation from lymphocytes). The mixture is centrifuged at 12,000g for 15min (5min for the isolation from lymphocytes) at 4°C. The uppermost aqueous layer, which contains the sample RNA, is transferred to a new RNAse-free tube (see Note 21). The extraction is performed at least twice with phenol/chloroform/isoamyl alcohol using RNAse-free plastic tubes. 

The volume of the aqueous solution is measured, and one-tenth of the volume sodium acetate solution is added. An equal volume of acid phenol/chloroform is added as described in step 3, and the mixing, incubation on ice, and centrifugation are repeated once (see Note 22). The upper aqueous RNA-containing phase is transferred to a new RNAse-free tube, and the volume is measured. 

Centrifugation tubes of these materials wiU withstand phenol/chloroform solutions and 12,000 g forces. 

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. 

Add equal amount of phenol/chloroform solution, mix well, and centrifuge at 17,000 x for 3 min. 

Phenol Chloroform Isoamyl Alcohol 25 24 1 saturated with 10 mM Tris-HCl, pH 8.0, 1 mM EDTA Store at 4°C. 

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. 

The amplified cDNA template can be purified via phenol-chloroform-isoamyl alcohol extraction or with commercially available column kit. However, it was found that this purification step is not necessary for the following procedures. 

Venous blood (8 mL) was collected into 15-mL tubes containing 50 mmol/L disodium EDTA, and genomic DNA was extracted by the standard (phenol/chloroform) method. The genotype frequencies for GSTPl were determined by polymerase chain reaction (PCR)/RFLP-based methods using DNA extracted from peripheral lymphocytes. 

Extract the sample with phenol/chloroform (11) once, then with chloroform once. 

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