Luciferase solution preparation

Contaminating CO2. The ubiquitous presence of CO2 causes a great difficulty to the experiment. Normal atmosphere contains 0.03% CO2, and a 5-6 ml portion of pure water equilibrated with atmosphere contains 0.06 pmol of CO2. However, the amount of CO2 in buffers and luciferase solutions is much greater. For example, 5 ml of freshly prepared Tris-HCl buffer, pH 7.8, contained 0.13 pmol of CO2 after 20 min degassing, and the value increased to 0.23 pmol when the same buffer was tested one week later (Shimomura et al., 1977). It is strongly advised to eliminate all nonessential CO2 sources from the experimental environment. 

Prepare Luciferase Solution. Keep in dark and incubate at room temperature for 15 min. 

Plasmid DNA solution prepare DNA solution e.g. luciferase reporter plasmid or eGFP plasmid, at a concentration of 12 pg DNA/ml by dilution of the stock solution with a serum- and supplement-free medium (e.g., RPMl 1640). 

Reporter DNA stock solution Prepare 1 mL of a DNA reporter stock solution by combining 300 pL of p53-Firefly luciferase (FL), 300 pL of SXTCF-Renilla luciferase (RL), 300 pL of Elkl-Gal4, 60 pL UAS-Cypridina luciferase (GL), and 40 pL of water to achieve a flnal 5 5 5 1 ratio of reporters. The final total DNA concentration of this stock solution is 0.96 mg/ mL. 

Luciferase-catalyzed luminescence of luciferin. Odontosyllis luciferin emits light in the presence of Mg2+, molecular oxygen and luciferase. The relationship between the luminescence intensity and the pH of the medium shows a broad optimum (Fig. 7.2.8). The luminescence reaction requires a divalent alkaline earth ion, of which Mg2+ is most effective (optimum concentration 30 mM). Monovalent cations such as Na+, K+, and NH have little effect, and many heavy metal ions, such as Hg2+, Cu2+, Co2+ and Zn2+, are generally inhibitory. The activity of crude preparations of luciferase progressively decreases by repeated dialysis and also by concentrating the solutions under reduced pressure. However, the decreased luciferase activity can be completely restored to the original activity by the addition of 1 mM HCN (added as KCN). The relationship between the concentration of HCN and the luciferase activity is shown in Fig. 7.2.9. Low concentrations of h and K3Fe(CN)6 also enhance luminescence, but their effects are only transient. 

The solution is dialyzed against the same buffer using a hollow fiber assembly, and then added onto a column of Affi-Gel Blue (50-100 mesh, 2 x 15 cm, Bio-Rad) prepared with the same buffer. The column is washed with the same buffer. Then luciferase is eluted with 50 mM Tris-HCl, pH 8.5, containing 5mM EDTA, 3 mM DTT, and 0.5 M NaCl (Hastings and Dunlap, 1986, state that it may be preferable to omit the Affi-Gel step because of difficulties encountered). 

The next day, infect the HT-29 cells with luciferase or mock retroviral particles. A 2.5 ml solution containing retroviral particles is either prepared fresh or obtained from a frozen aliquot (see above). Prepare polybrene as 100 x stock solution (0.8 mg/ml) in 1 x PBS (can be stored at —20°C). Add polybrene to the tubes containing the retroviral particles at a final concentration of 8 pg/ml. Aspirate the medium from the HT-29 cells in the two T25 flasks. Initiate the infection by adding 2.5 ml of the solutions containing the luciferase or mock retroviral particles with polybrene to both flasks. 

Before measurements one vial of bacteria was diluted by 500 pL 1.5 % NaCl. 20 pL of bacteria solution was added to 1 mL of 3 % NaCl and the control light intensity was recorded using the bioluminometer after 15 min period of incubation. The measurements were repeated with 1 mL 3 % NaCl prepared on water samples and experimental light intensity was measured. The effect of sample water on coupled enzyme system bioluminescence was estimated by the bacterial (BI) or luciferase (LI) index using the following formula BI = If lao LI = If I. ... 

To demonstrate ricin detection, a series of concentrations of ricin solutions are prepared from a stock solution of 35 pM. Then, 2 pL of each ricin sample is added into 6 pL of the reaction solution in the reaction chamber in Figure lb. The volume of the feeding solution remained at 80 pL. For the positive controls in the same device, 2 pL of water is added. The negative controls contain no luciferase vector, providing with the background signal. 

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