By Ray Wu, Lawrence Grossman, Kivie Moldave
The looks of one other quantity in that first-class sequence, equipment in Enzymology, is usually a reason for appreciation should you desire to effectively perform a selected process or arrange an enzyme or metabolic intermediate with no the tiresome prospect of looking through unusual literature and maybe determining an unproven process which isn't simply reproduced.
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Extra info for Recombinant DNA Part B
G. Barrio, N. J. Leonard, T. E. England, and O, C. Uhlenbeck, Biochemistry 1 7 , 2 0 7 7 ( 1 9 7 8 ) . METHODS IN ENZYMOLOGY, VOL. 100 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182000-9  R N A LIGASE WITH D N A SUBSTRATES 39 The enzyme mechanism proceeds via the formation of two covalent intermediates. 9 In a second step, the enzyme transfers the adenylyl group to the 5'-phosphate of the donor to form the structure Ado-5'PP5'-X, where P5'-X represents the donor.
I. Tikchonenko, E. V. Karamov, B. A. Zavizion, and B. S. Naroditsky, Gene 4, 195 (1978). k M. Hsu, and P. Berg, Biochemistry 17, 131 (1978). H. Belle Isle, unpublished results, 1981. 32 ENZYMES IN RECOMBINANT D N A  should be performed under the recommended standard assay conditions especially in regard to pH, ionic strength, and divalent cation concentration. The amount of glycerol introduced into the assay should be kept below 5% (v/v), and prolonged incubation with high enzyme-to-DNA ratios should be avoided.
Hydroxyapatite Chromatography. 9 cm diameter x 20 cm) as described. 19 RNA ligase elutes at approximately 60-80 mM KPi. Fractions containing activity were partially concentrated by pressure filtration using a PM-10 membrane. 5), 1 mM DTT further concentrated the enzyme. The dialysis medium was changed four times at 30-min intervals to remove Pi from the sample. The medium was then changed to buffer III twice for 30 min to return MgCI2 to the solution (fraction VI, 11 ml). Phosphate must be removed from the solution before Mg(II) is added to avoid formation of a precipitate.
Recombinant DNA Part B by Ray Wu, Lawrence Grossman, Kivie Moldave