Methods in Molecular Biology • 16 Enzymes of Molecular Biology

338 Maunders The above procedures refer to CLAP, since it is very difficult to remove BAP after reaction. A newly available bact ...

Alkaline Phosphatase 339 References Fernley, H. N. (1971) Mammalian alkaline phosphatases, in The Enzymes, vol. 4, 3rd ed. (Boy ...

340 Maunders Engstrom, L. (1961) Studies on calf intestinal alkaline phosphatase. 1. Chro- matographic purification, microheter ...

Alkaline Phosphatase 341 Barman, T. E. (1969) Alkaline phosphatase, in The Enzyme Handbook, vol. 2, Springer-Verlag, Berlin, pp ...

CHAPTER 20 Polynucleotide Kinase (EC 2.7.1.78) Martin J. Maunders Introduction The enzyme polynucleotide kinase (PNK, or ATP:5' ...

344 Maunders The bacteriophage polynucleotide kinases have similar properties, that from T4-infected E. coli having been the mos ...

Kinase 345 The associated 3'-phosphatase activity causes the hydrolysis of the 3'-phosphate group of a variety of substrate mole ...

346 Maunders The following K m values have been determined for T2 polynucleo- tide kinase: 14 lxM for ATP, 15 ~$/for UTP, 33 lxM ...

Kinase 347 3.6. Activators The activity of polynucleotide kinase is stimulated by sodium chlo- ride, potassium chloride, and pol ...

348 Maunders 3.8. Sulfhydryl Reagents Sulfhydryl reagents are essential for the action of polynucleotide kinase (18), whose acti ...

Kinase 349 End-labeling allows the quantification of the termini, the enzyme often being used in conjunction with alkaline phosp ...

350 Maunders tions can be varied to give the optimal reaction with a particular sub- strate. For example, when kinasing blunt or ...

Kinase 351 7.5M Ammonium acetate. Ethanol. TE buffer: 10 mM Tris-HC1, pH 8.0, 1 mM EDTA. 11.24% PEG 8000. 1 mM ADP solution (fo ...

352 Maunders Incubate at 37°C for 30 min. Terminate the reaction by adding 2 pL of 500 mM EDTA, pH 8.0. Extract, purify, and me ...

Kinase 353 If the oligonucleotide is not labeled highly enough, add another 10 U of enzyme and incubate for a further 30 min. P ...

354 Maunders Weiss, B., Live, T. R., and Richardson, C. C. (1968) Enzymatic breakage and joining of deoxyribonucleic acid. V. E ...

Kinase 355 Okazaki, R., Hirose, S., Okazaki, T., Ogawa, T., and Kurosawa, Y. (1975) Assay of RNA-linked nascent DNA pieces with ...

356 Maunders the cyclobutane pyrimidine dimer and cis and trans dichlorodiammine plati- num II examples. Nucleic Acids Res. 9, 4 ...

Index A AatI, 130, see also Restriction enzymes AatlI, 130, see also Restriction enzymes AccI, 130, see also Restriction enzymes ...

358 Index iminopeptidase, 319 L-pyroglutamyl peptide hydrolase, 321 loss of serine and threonine, 320 loss of tryptophan, 320 me ...