Table 1.

Protocol for Processing the Plate-Based Assay for Nonspecific DNA Nicking

1. 10-µl reactions are set up in 384-well plates that are kept at 4°C.

2. Plates are sealed, briefly spun, then incubated at 37°C for 90 min.

3. Reactions are stopped by placing in a 65°C oven for 10 min to inactivate integrase.

4. Plates are spun again and placed on the queue for a real-time PCR systema.

5. The machine is programmed for a single denaturation at 95°C for 1 min and renaturation at     65°C for 1 min, followed by 2 min 59 secb at 65°C during which fluorescence is read 21 times     in the absolute quantitation mode (no cycling is performed).

6. The data are exported as a Microsoft Excel filec.

7. The data are copied and pasted into a pre-templated Excel filed that automatically averages the     final 10 reads for each well, calculates the control data, and displays the results on bar graphs.

^aThe protocol was developed using an Applied Biosystems 7900HT Real-Time PCR System.

^bWhen a data collection phase of precisely 3 min was tried, the machine started another pass across the plate and read one of the outer columns of wells an extra time.

^cIn the absolute quantitation mode with this system, each well generates 31 rows of data or information; thus, the output for a 384-well plate is an Excel file with > 10,000 rows.

^dAvailable on request.