Phenols phenol chloroformate

In contrast to tissues, tissue culture cells are readily lysed with detergent [11]. Adherent cells from a culture plate are first scraped using a rubber policeman into a small volume of phosphate-buffered saline (137 mMNaCl, 2.7 mM KC1, 4.3 mM Na2HP04, 1.4 mM KH2P04, pH 7.3) and harvested by centrifugation at 1500 x g for 10 minutes at 4°C. The cellular pellet is resuspended in ice-cold TE so that 1 mL contains 100 million cells. After the addition of 10 volumes of freshly prepared digestion buffer (10 mM Tris-Cl, pH 8, 0.05 EDTA, pH 8, 0.5% Sarcosyl, and 100 pg/mL proteinase K), the sample is incubated at 50°C for 3 hours. DNA is recovered by ethanol precipitation after extraction with phenol, phenol-chloroform, and chloroform as described earlier. 

Phenol, Phenol/chloroform extractions (Doing this carefully is very important)... 

Although 4-hydroxybenzaldehyde can be made by the saligenin route, it has been made historically by the Reimer-Tiemann process, which also produces sahcylaldehyde (64). Treatment of phenol with chloroform and aqueous sodium hydroxide results in the formation of benzal chlorides, which are rapidly hydrolyzed by the alkaline medium into aldehydes. Acidification of the phenoxides results in the formation of the final products, sahcylaldehyde and 4-hydroxybenzaldehyde. The ratio of ortho and para isomers is flexible and can be controlled within certain limits. The overall reaction scheme is shown in Figure 1. Product separation is accomphshed by distillation, but this process leads to environmental problems because of the quantities of sodium chloride produced. 

Phenols. Phenols are unreactive toward chloroformates at room temperature and at elevated temperatures the yields of carbonates are relatively poor (< 10%) in the absence of catalysis. Many catalysts have been claimed in the patent Hterature that lead to high yields of carbonates from phenol and chloroformates. The use of catalyst is even more essential in the reaction of phenols and aryl chloroformates. Among the catalysts claimed are amphoteric metals or thek haUdes (16), magnesium haUdes (17), magnesium or manganese (18), secondary or tertiary amines such as imidazole (19), pyridine, quinoline, picoline (20—22), heterocycHc basic compounds (23) and carbonamides, thiocarbonamides, phosphoroamides, and sulfonamides (24). 

Although less common, azeotropic mixtures are known which have higher boiling points than their components. These include water with most of the mineral acids (hydrofluoric, hydrochloric, hydrobromic, perchloric, nitric and sulfuric) and formic acid. Other examples are acetic acid-pyridine, acetone-chloroform, aniline-phenol, and chloroform-methyl acetate. 

The Reimer-Tiemann reaction of phenol with chloroform, in the presence of 50% NaOH, gives a much higher ratio of para to ortho hydroxy benzaldehyde when polyethylene glycol is used as compared to the established procedure without any addition of a hydrotope. A para to ortho ratio of 1 1 has been realized (Neumann and Sasson, 1986). 

The well-known reaction between phenol and chloroform in the presence of concentrated sodium hydroxide is amenable to dramatic changes with cyclodextrins. -cyclodextrin, immobilized with epichlorohydrin, seems to give 100 % selectivity for para... 

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. 

The plasmid p/wc-A60 is digested with Ilpal for synthesis of luciferase mRNA with a S -terminal 60-nt poly(A) tail. The RNAs are synthesized in 200-pl reaction mixtures incubated for 2 h at 37° using conditions described previously. Reaction mixtures are further treated with 3 U of DNase RQ1 (Promega) for 20 min at 37°, extracted with phenol and chloroform, and... 

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. 

Proton donors alcohols, carboxylic acids, phenols, and chloroform Proton acceptors amines, ethers, sulfoxides, amides, esters, and alcohols... 

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. 

THE REIMER-TIEMANN SYNTHESIS SALICYLALDEHYDE FROM PHENOL AND CHLOROFORM 1... 

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... 

Reimer-Tiemann reaction org chem Formation of phenolic aldehydes by reaction of phenol with chloroform in the presence of an alkali. rTm-or te-m3n re.ak-shon Reinecke s salt analychem (Ntf3)2Cr(SCN)4 NH4 H2O A reagent to detect mercury (gives a red color or a precipitate), and to isolate organic bases (such as proline or histidine). rTn-o-kez, s6lt ... 

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. 

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