Nucleic Acids and Protein Synthesis Part E by Kivie Moldave, Lawrence Grossman

By Kivie Moldave, Lawrence Grossman

The significantly acclaimed laboratory usual, Methods in Enzymology, is among the so much hugely revered courses within the box of biochemistry. considering that 1955, every one quantity has been eagerly awaited, often consulted, and praised via researchers and reviewers alike. The sequence includes a lot fabric nonetheless correct this present day - really an important e-book for researchers in all fields of existence sciences

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Extra resources for Nucleic Acids and Protein Synthesis Part E

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6%, respectively. 5% (w/w) ; the mixture is stirred for 2 hours, and then centrifuged at 4000 g for 10 minutes. The resulting clear, yellow, upper polyethylene glycol phase is recovered, and the turbid, lower dextran phase containing the bulk of the nucleic acids is discarded. The polyethylene glycol phase (fraction II) can be stored at - 15° for 2 months without loss of activity. Step 3. Flotation of Polyethylene Glycol. 4, containing 10 mM 2-mercaptoethanol and 2 mM EDTA for a total of 6 hours.

H. Sells, Anal. Biochem. 22, 195 (1968). [5] PURIFICATION OF DNA POLYMERASE 41 perature. 1 mM 2-mercaptoethanol. Except where indicated, all operations are carried out at 0-4 ° in a cold room. Micrococcus luteus (ATCC No. , Baltimore, Maryland) and harvested during late logarithmic phase after 24 hours of growth (5-8 g/liter). The cells are available spray dried from Miles Laboratories, Elkhart, Indiana, and from the Grain Processing Company, Muscatine, Iowa. 0 and can be stored at --70 ° without loss of DNA polymerase activity.

2 Our investigations into the residual polymerase activity in p o l A - c e i l s have resulted in the isolation of two other D N A polymerases which we call polymerase I I and polymerase I I I , in order of discovery. The three enzymes bear some similarities to the correspondingly numbered D N A polymerases from Escherichia coli, ~-6 but differences do exist, and thus extrapolation should be done with caution. Assay Method Principle. The three enzymes catalyze the covalent addition of nucleotides to a D N A primer under the direction of a D N A template using the four common deoxyribonucleoside 5'-triphosphates as substrate.

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