By Ray Wu, Lawrence Grossman, Kivie Moldave
The looks of one other quantity in that first-class sequence, equipment in Enzymology, is often a reason for appreciation in the event you desire to effectively perform a specific procedure or organize an enzyme or metabolic intermediate with no the tiresome prospect of looking through unexpected literature and maybe picking an unproven process which isn't simply reproduced.
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Additional resources for Recombinant DNA Part B
A. Myers, G. A. Weiss, R. J. Roberts and K. Murray, J. Mol. Biol. 114, 433 (1977). 55 S. Sato, C. A. Hutchison III, and J. I. Harris, Proc. Natl, Acad. Sci. A. 74, 542 (1977). 9 Hence, the order of addition of components to the reaction mixture should place the enzyme last, at which point the reaction is deemed to have started. Stopping Reactions Restriction endonuclease reactions can be stopped by one of several different methods. The method chosen depends upon the subsequent use of the DNA products.
4 is attained and the enzyme has been concentrated to 6-10 mg/ml, the solution is clarified by centrifugation and an equal volume of glycerol is added (fraction VII, 7 ml). The enzyme is stored at -20 °. , end-labeling RNA with [5'-32p]prCp, fraction VI is satisfactory. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate shows that greater than 95% of the protein migrates as the unadenylylated enzyme. A portion of the enzyme elutes from the final Affi-Gel Blue column in the adenylylated form.
16 D. M. Hinton and R. I. Gumport, Nucleic Acids Res. 7, 453 (1979). 17 M. M. McCoy and R. I. Gumport, Biochemistry 19, 635 (1980). 18 D. M. Hinton, C. A. Brennan, and R. I. Gumport, Nucleic Acids Res. 10, 1877 (1982). 19 We h a v e used cells infected with bacteriophage T4 bearing a m b e r mutations in either gene 43 or 45 and h a v e obtained satisfactory results with both. 19,2° The gene 45 mutant has the advantage of allowing purification of T4 D N A p o l y m e r a s e as well as T4 polynucleotide kinase and T4 D N A ligase f r o m the cell extract.
Recombinant DNA Part B by Ray Wu, Lawrence Grossman, Kivie Moldave