2-amino-2-(hydroxymethyl)propane-1,3-diol;hydrochloride

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 10 mM Tris-HCl (pH 7.8), 0.1 mM DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)), 0.25 mM acetyl CoA, 5 mM cadaverine, enzyme solution

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: By spectrophotometric analysis, the elutions in 100 ul of 10 mM Tris HCl at pH 9.5 yielded 30 μg (A260/A280 of 1.78) of DNA for the bimodal urea-histidine-propionate IE particles and less than 2 μg of DNA for the urea-histidine IE particles. No DNA was detected on analysis of the eluent from the urea-histidine IE particles, by gel electrophoresis.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 45.375 g of Tris base (Sigma; T1503) to be dissolved in 200 ml of distilled water, adjust the pH to 7.5 with 12N HCl and then make up to 250 ml

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To 300 μl of a solution comprising 10 mM Tris-HCl (pH 7.5), 6 mM MgCl2 and 10 mM NaCl, there was added 400 μg of pCDV1 [Okayama & Berg: Mol. Cell. Biol., 3, 280 (1983)] and, after further addition of 500 units of KpnI, the reaction was carried out at 37° C. for 6 hours, whereby the plasmid was cleaved at the KpnI site. The DNA recovered byphenol-chloroform extraction followed by ethanol precipitation. About 200 μg of the KpnI-cleaved DNA was added to 200 μl of a solution prepared by adding dTTP in a concentration of 0.25 mM to TdT buffer and, after further addition of 81 units of TdT (P-L Biochemicals), the reaction was carried out at 37° C. for 11 minutes, whereby a poly(dT) chain (about 67 dT residues) was added to each 3' end of the KpnI cleavage site of pCDV1. About 100 μg of the poly(dT) chain-tailed pCDV1 DNA was recovered from the solution by ethanol precipitation following phenol-chloroform extraction. The DNA was added to 150 μl of a solution comprising 10 mM Tris-HCl (pH 7.5), 6 mM MgCl2 and 100 mM NaCl and, after further addition of 360 units of EcoRI, the reaction was carried out at 37° C. for 2 hours. The reaction mixture was treated by the LGT method, and a DNA fragment of about 3.1 kb was recovered. About 60 μg of the poly(dT) chain-tailed pCDV1 was thus obtained. The DNA was dissolved in 500 μl of a solution comprising 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA, the solution was incubated at 65° C. for 5 minutes and the cooled with ice, and 50 μl of 5M NaCl was added. The mixture was subjected to oligo(dA)-cellulose column (Collaborative Research) chromatography. Molecules having a sufficient poly(dT) chain length were adsorbed on the column and they were eluted with a solution comprising 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA to give 27 μg of the poly(dT) chain-tailed pCDV1 (hereinafter referred to as vector primer). was added 400 μg of pCDV1 [Okayama & Berg: Mol. Cell. Biol., 3, 280 (1983)] The DNA recovered byphenol-chloroform extraction followed by ethanol precipitation About 200 μg of the KpnI-cleaved DNA was added to 200 μl of a solution prepared the reaction was carried out at 37° C. for 11 minutes whereby a poly(dT) chain (about 67 dT residues) was added to each 3' end of the KpnI cleavage site of pCDV1 About 100 μg of the poly(dT) chain-tailed pCDV1 DNA was recovered from the solution by ethanol precipitation phenol-chloroform extraction The DNA was added to 150 μl of a solution after further addition of 360 units of EcoRI the reaction was carried out at 37° C. for 2 hours The reaction mixture was treated by the LGT method a DNA fragment of about 3.1 kb was recovered

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To 300 μl of a solution comprising 10 mM Tris-HCl (pH 7.5), 6 mM MgCl2 and 10 NaCl, there was added 400 μg of pCDV1 [Okayama & Berg: Mol. Cell. Biol., 3, 280 (1983)] and , after furthere addition of 500 units of KpnI, the reaction was carried out at 37° C. for 6 hours, whereby the plasmid was cleaved at the KpnI site. The DNA was recovered by phenol-chloroform extraction and ethanol precipitation. About 200 μg of the KpnI-cleaved DNA was added to 200 μl of a solution prepared by adding dTTP in a concentration of 0.25 mM to a buffer (hereinafter abbreviated as TdT buffer) comprising 40 mM sodium cacodylate, 30 mM Tris-HCl (pH 6.8), 1 mM CaCl2 and 0.1 mM dithiothreitol (hereinafter abbreviated as DTT) and, after further addition of 81 units of terminal deoxy-nucleotidyl transferase (hereinafter abbreviated as TdT) (P-L Biochemicals), the reaction was carried out at 37° C. for 11 minutes, whereby a poly(dT) chain comprising about 67 dT residues was added to each KpnI cleavage site 3' end of pCDV1. About 100 μg of the poly(dT) chain-added pCDV1 DNA was recovered from the above reaction mixture by phenol-chloroform extraction and ethanol precipitation. The DNA was added to 150 μl of a buffer comprising 10 mM Tris-HCl (pH 7.5), 6 mM MgCl2 and 100 mM NaCl, 360 units of EcoRI was further added, and the reaction was carried out at 37° C. for 2 hours. The reaction mixture was treated by the LGT method, and a DNA fragment of about 3.1 kb was recovered. Thus was obtained about 60 μg of the poly(dT) chain-tailed pCDV1. The DNA was dissolved in 500 μl of a solution comprising 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA, the solution was incubated at 65° C. for 5 minutes and then cooled with ice, and 50 μl of 5M NaCl was added. The mixture was subjected to oligo(dA)-cellulose column (Collaborative Research) chromatography. Molecules having a sufficient poly(dT) chain length were adsorbed on the column and they were eluted with a solution comprising 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. Thus was obtained 27 μg of the poly(dT) chain-tailed pCDV1 (hereinafter abbreviated as vector primer). was added 400 μg of pCDV1 [Okayama & Berg: Mol. Cell. Biol., 3, 280 (1983)] The DNA was recovered by phenol-chloroform extraction and ethanol precipitation About 200 μg of the KpnI-cleaved DNA was added to 200 μl of a solution prepared the reaction was carried out at 37° C. for 11 minutes was added to each KpnI cleavage site 3' end of pCDV1 About 100 μg of the poly(dT) chain-added pCDV1 DNA was recovered from the above reaction mixture by phenol-chloroform extraction and ethanol precipitation The DNA was added to 150 μl of was further added the reaction was carried out at 37° C. for 2 hours The reaction mixture was treated by the LGT method a DNA fragment of about 3.1 kb was recovered

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Nuclei were isolated from suspension cultured cells according to the procedure of Lawton and Lamb, Mol. Cell Biol. 7, 335-341 (1987). Isolated nuclei were incubated in 0.1M ammonium sulfate, 4 mM MgCl2, 0.3 mM phosphocreatine, 0.15 mg/ml creatine phosphokinase, 1 U/ml RNasin (Promega), 0.5 mL each ATP, CTP, GTP, and 2 mCi/ml 32P-UTP (3000 Ci/mmol). The standard reaction volume was 30 ml containing 15 ml nuclei. The reaction mixture was incubated 30 minutes at 30° C. Three units RQ1 DNAse (Promega) and 2 ml 20 mM CaCl2 were added and the reaction mix incubated at 26° C. for 5 minutes. A mixture containing 1.5 ml proteinase K (2 mg/ml), 9 ml 10×SET (1×SET is 0.5% SDS, 5 mM EDTA, 10 mM Tris-HCl pH 7.4) and 50 mg yeast tRNA was added and the reaction was continued at 37° C. for 30 minutes. Labelled RNA was isolated by acid guanidinium/phenol/chloroform extraction. The assay consistently yielded 2-5×106CPMs. Nuclei were isolated from suspension cultured cells containing 15 ml the reaction mix incubated at 26° C. for 5 minutes was added the reaction was continued at 37° C. for 30 minutes

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The starting yeast is Saccharomyces cerevisiae AH22 [a leu 2 his4 can 1 (Cir+)], which has been deposited at Fermentation Research Institute, Agency of Industrial Science and Technology, Japan under Budapest Treaty as "FERM BP-312". The starting yeast is inoculated in YPD medium (100 ml) containing 2% polypeptone, 1% yeast extract and 2% glucose, and the mixture is incubated at 30° C. overnight, and thereafter, the cells are collected by centrifugation. The cells are washed with sterilized water (20 ml) and suspended in a solution (5 ml) of 1.2M sorbitol and 100 μg/ml zymolyase-60,000 (manufactured by Seikagaku Kogyo K. K., Japan), and the suspension is allowed to stand at 30° C. for about 30 minutes to give spheroplast. The spheroplast thus prepared is washed with 1.2M sorbitol solution three times, and then suspended in a solution (0.6 ml) of 2M sorbitol, 10 mM CaCl2 and 10 mM Tris-HCl (pH 7.5). The suspension thus prepared is divided into a small test tube in a volume of 60 μl. To the suspension is added the recombinant plasmid pONY-S1 (5 μg) as prepared in the above (3). After mixing well, 0.1M CaCl2 (3 μl) is added thereto in a final concentration of 10 mM CaCl2, and the mixture is allowed to stand at room temperature for 5 to 10 minutes. To the resulting mixture is added each 1 ml of 20% polyethylene glycol 4,000, 10 mM CaCl2 and 10 mM Tris-HCl (pH 7.5), and the mixture is allowed to stand at room temperature for about 20 minutes. The resulting mixture (each 0.2 ml) is added to a medium (10 ml) [which consists of 22% sorbitol, 2% glucose, 0.7% yeast nitrogen base amino acid, 2% YPD, 20 μg/ml histidine, and 3% agar], which is kept at a constant temperature of 45° C. After gently mixing, the mixture is added in a layer onto a plate of minimal medium containing 1.2M sorbitol which is previously prepared and consists of 0.7% yeast nitrogen base amino acid, 2% glucose, 20 μg/ml histidine and 2% agar and is set thereon. The plate is incubated at 30° C. to give colony of a leucine-non-requiring yeast. The colony is incubated in a Burkholder minimal medium supplemented with histidine (20 μg/ml) [cf. Tohe, A. et al., J. Bacteriol., 113, 727-738 (1973)] to give the desired transformed yeast: Saccharomyces cerevisiae YpONY-S1. containing 2% polypeptone, 1% yeast extract the cells are collected by centrifugation The cells are washed with sterilized water (20 ml) to stand at 30° C. for about 30 minutes to give spheroplast The spheroplast thus prepared is washed with 1.2M sorbitol solution three times The suspension thus prepared is divided into a small test tube in a volume of 60 μl To the suspension is added the recombinant plasmid After mixing well to stand at room temperature for 5 to 10 minutes to stand at room temperature for about 20 minutes The resulting mixture (each 0.2 ml) is added to a medium (10 ml) [which is kept at a constant temperature of 45° C After gently mixing the mixture is added in a layer onto a plate of minimal medium is previously prepared is incubated at 30° C.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The starting yeast is Saccharomyces cerevisiae AH 22 [a, leu 2, his 4, can 1 (Cir+)], which has been deposited at Fermentation Research Institute, Agency of Industrial Science and Technology, Japan under Budapest Treaty as "FERM BP-312". The starting yeast is inoculated in YPD medium (100 ml) consisting of 2% polypeptone, 1% yeast extract and 2% glucose, and the mixture is incubated at 30° C. overnight, and thereafter, the cells are collected by centrifugation. The cells thus collected are washed with sterilized water (20 ml), suspended in a solution (5 ml) of 1.2M sorbitol and Zymolyase-60,000 (manufactured by Seikagaku Kogyo K. K., Japan, 100 μg/ml), and the suspension is allowed to stand at 30° C. for 30 minutes to yield spheroplasts. The spheroplasts thus prepared are washed with 1.2M sorbitol solution three times, and then suspended in a solution (0.6 ml) of 2M sorbitol, 10 mM CaCl2 and 10 mM Tris-HCl (pH 7.5). The suspension thus prepared is divided into a small test tube in a volume of 60 μl. To the suspension is added the solution of the recombinant plasmid pONYGB2 (10 μg) as prepared above (3). After mixing well, 0.1M CaCl2 (3 μl) is added thereto in a final concentration of 10 mM CaCl2, and the mixture is allowed to stand at room temperature for 5 to 10 minutes. To the resulting mixture is added each 1 ml of a solution of 20% polyethylene glycol 4,000, 10 mM CaCl2 and 10 mM Tris-HCl (pH 7.5), and the mixture is allowed to stand at room temperature for about 20 minutes. The resulting mixture (each 0.2 ml) is added to a medium (10 ml) consisting of 22% sorbitol, 2% glucose, 0.7% yeast nitrogen base amino acid, 2% YPD, 20 μg/ml histidine and 3% agar, which is kept at a constant temperature of 45° C. After gently mixing, the mixture is added in a layer onto a plate of minimal medium containing 1.2M sorbitol which is previously prepared and consists of 0.7% yeast nitrogen base amino acid, 2% glucose, 20 μg/ml histidine and 2% agar and is set thereon. The plate is incubated at 30° C. to yield colonies of non-leucine requiring yeast. A colony is incubated in a BurkHolder minimal medium supplemented with histidine (20 μg/ml) [cf. Tohe, A. et al; J. Bacterol., 113, 727-738, 1973] to give the desired transformed yeast: Saccharomyces cerevisiae YGBS. extract the cells are collected by centrifugation The cells thus collected are washed with sterilized water (20 ml) to stand at 30° C. for 30 minutes to yield spheroplasts The spheroplasts thus prepared are washed with 1.2M sorbitol solution three times The suspension thus prepared is divided into a small test tube in a volume of 60 μl To the suspension is added the solution of the recombinant plasmid pONYGB2 (10 μg) After mixing well to stand at room temperature for 5 to 10 minutes to stand at room temperature for about 20 minutes The resulting mixture (each 0.2 ml) is added to a medium (10 ml) is kept at a constant temperature of 45° C After gently mixing the mixture is added in a layer onto a plate of minimal medium is previously prepared is incubated at 30° C.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The thus prepared solution of the test compound was mixed with a solution containing 50 mM of Tris-HCl buffer (pH 8.3), 8 mM of MgCl2, 30 mM of NaCl, 50 mM of dithiothreitol (made by Wako Pure Chemical Industries Ltd.), 0.2 mM of thymidine-5'-triphosphate (made by Pharmacia K. K.) and 6 U/ml of reverse transcriptase derived from M-MLV (made by Pharmacia K. K.), and preincubated at 37° C. for 30 minutes. After there was added thereto 10 μg/ml of polyadenylic acid (made by PL Biochemicals Co., Ltd.), 0.01 U/ml of oligodeoxy thymidylic acid (made by Pharmacia K. K.) and 10 μCi/ml of [methyl-3H] thymidine-5'-triphosphate (made by Amersham Japan Co., Ltd., 47 Ci/mmol) to give a reaction mixture, the mixture was further incubated at 37° C. for 30 minutes, followed by cooling with ice to stop the reaction. The thus prepared solution of the test compound was mixed with a solution After there was added

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The first solid phase, i.e. the DNA-binding beads, are preferably Magprep Silica HS beads that are preferably diluted 1 in 3 in lysis buffer from a stock solution of 50 mg/ml to get in total 833.3 mg beads at a volume of 50 mL. Correspondingly, the second solid phase, i.e. the RNA-binding beads, are preferably Magprep Silica Basic beads that are preferably diluted 1 in 3 in lysis buffer from a stock solution of 50 mg/ml to get in total 833.3 mg beads at a volume of 50 mL. The kit preferably also comprises lysis buffer with Proteinase K, preferably in the form of 7 M Guanidine HCl, 50 mM Tris, 2% Tween (pH 7.0) and Proteinase K 20 mg/ml. The total volume of lysis buffer and the Proteinase K in the kit could be 110 mL with 95/100 parts lysis buffer and 5/100 parts Proteinase K. When extracting DNA and RNA from a sample 800 μL of lysis buffer and 40 μL of Proteinase K are added per extraction sample. The wash buffer, preferably 10 mM TrisHCl (pH 6.5) can be provided at a volume of 265 mL and wash buffer with DNase could be provided at a total volume of 75 ML and 98/100 parts of the wash buffer, 1/100 part of DNase at 1 U/μL and 1/100 part of DNase buffer. The kit preferably also comprises an elution buffer, such as 10 mM TrisHCl, 1 mM EDTA (pH 8.6) at a volume of 100 mL. that are preferably diluted 1 in 3 in lysis buffer from a stock solution of 50 mg/ml to get in total 833.3 mg that are preferably diluted 1 in 3 in lysis buffer from a stock solution of 50 mg/ml to get in total 833.3 mg When extracting DNA and RNA from a sample 800 μL of lysis buffer 40 μL of Proteinase K are added per extraction sample

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: First, a vector primer is synthesized. A vector, e.g. pcDV1, is treated with KpnI in an adequate solution such as a solution consisting of Tris-HCl buffer (e.g. pH 7.5, 10 mM), MgCl2 (e.g. 6 mM) and NaCl (e.g. 10 mM) to cut pcDV1 at KpnI site. The DNA is incubated with terminal deoxynacleotidyltransferase at an appropriate temperature (e.g. 37° C.) for an appropriate period (e.g. 20 minutes) in a solution consisting of Tris-HCl buffer (e.g. pH 6.8, 30 mM), sodium cacodylate (e.g. 140 mM), CoCl2 (e.g. 1 mM), dithiothreitol (e.g. 0.1 mM) and dTTP (e.g. 0.25 mM) to add about 60 thymidyl residues to the both 3' ends of the vector DNA. Then, the DNA is cut with EcoRI in a solution consisting of Tris-HCl buffer (e.g. pH 7.5, 10 mM), MgCl2 (e.g. 6 mM) and NaCl (e.g. 100 mM) The digested solution is fractionated by low-gelling-temperature agarose gel electrophoresis [Lars Wieslahder: Analytical Biochemistry, 98, 305 (1979), referred to as LGT method hereinafter] to recover a DNA fragment of about 3.1 Kb. Then, the DNA is dissolved in an NaCl or KCl hypertonic solution (e.g. 0.5M) and passed through a poly(dA) cellulose column to allow only vector primer molecules having poly(T) to be adsorbed on the column. Elution is carried out with water or a hypotonic salt solution such as 10 mM Tris-HCl buffer to isolate only the vector primer molecule with poly(T). First, a vector primer is synthesized A vector, e.g. pcDV1, is treated with KpnI in an adequate solution such as a solution (e.g. 20 minutes) to recover a DNA fragment of about 3.1 Kb Then, the DNA is dissolved in an NaCl or KCl hypertonic solution (e.g. 0.5M) Elution

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The Arthrobacter species KNK168 strain was subjected to shaking culture in the same manner as in Example 6 using (RS)-1-methylpropylamine as an enzyme inducer. The cells harvested from one liter of the culture were suspended in one liter of a 0.1 M Tris-hydrochloric acid buffer (pH 8.5), and 30 g of 1-(3-trifluoromethylphenyl)-2-propanone, 18 g of (R)-1-phenylethylamine, and 44 g of oleic acid were added thereto. The components were reacted at 30° C. for 40 hours with stirring. After the reaction, the resulting mixture was subjected to extraction and distillation in the same manner as in Example 6, to give 19.5 g of (R)-1-(3-trifluoromethylphenyl)-2-aminopropane. The optical purity was 100% ee, and the yield was 65%. were added The components were reacted at 30° C. for 40 hours After the reaction the resulting mixture was subjected to extraction and distillation in the same manner as in Example 6