( direction. In the presence ofpyrophosph...)DNA Quantitation in the LMax Microplate Luminometer (MaxLine Application Note #37)

The transphosphorylation reaction is catalyzed by the enzyme nucleoside diphosphate kinase (NDPK). In this reaction, the terminal phosphate of the dNTP is transferred to ADP to form ATP. The ATP is then measured using a luciferin/ luciferase reagent to produce light in proportion to the amount of ATP which, in turn, is proportional to the amount of dsDNA.

MATERIALS
1. Lmax microplate luminometer (Molecular Devices Corp.)

2. DNAQuant DNA Quantitation System, Product No. K4000; Promega Corporation. 608-277-2516.

3. Solid white microplates; e.g. CorningCostar Cat. No. 3912, Tel: 1800492 1110)

PREPARATION OF REAGENTS AND STANDARDS
The instructions supplied with the kit (Promega Technical Bulletin No. 278) werefollowed, except for minor modifications to accommodate a 96-well format.
1. The enzyme and buffer solutions were thawed and kept on ice.

2. TE buffer (10 mM Tris and 1 mM EDTA in sterile, deionized water, pH adjusted to 7.4) was sterilized by filtration through an 0.2 m filter.

3. The luciferin/luciferase (ENLITEN L/L Reagent) was reconstituted by adding 12 mL of L/L Reconstitution Buffer to the vial, gently inverting several times and allowing to stand at room temperature for at least 30 min.

4. The Master Mix was prepared immediately before use by adding the following components in the order stated: 465 L DNA quantitation buffer solution, 15 L sodium pyrophosphate, 3 L NDPK enzyme solution, 27 L sterile, deionized water, 30 L T4 DNA polymerase.

5. The DNA standards were prepared by first diluting 50 L DNA stock standard (5 ng/mL; supplied in the kit) with 450 L TE buffer, followed by serial 1-to-2 dilutions i
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