Nuclease Test (OpenVent polymerase, PCR Master Mix, DNA loading dye)
Stephane Fadanka, Nadine Mowoh
Abstract
Quality control and nuclease test involves incubating the enzymes or reagents in reconstituted recombination assays to eliminate the possibility of relevant protein or nucleic acid contaminants that may interfere with recombination assays and lead to misinterpretation of experimental data. Here batch by batch quality controls are done for each new product before it is released to the market.
This protocol describes the steps in carrying out Nuclease test for:
- OpenVent Enzyme
- PCR Master Mixes - All formulations (Wet and Dry)
- 6x DNA loading dye
Before start
Clean and disinfect all work surfaces with a 1:10 dilution of bleach followed by 70% alcohol.
Make sure all resources needed are prepared.
Steps
Nuclease activity
Pipetting
- Pipette the following reagents into 0.2 mL PCR reaction tubes as shown in the table below, while working on ice.
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| Component | Test Sample (Enzyme and PCR Master mix) | Negative control | Positive control | Test Sample (DNA loading dye) | |
| Enzyme specific buffer | 2 µl | 2 µl | 2 µl | 2 µl | |
| Test sample | 5 µg | / | / | 5 µg | |
| Plasmid DNA | 1 µg | 1 µg | 1 µg | 1 µg | |
| DNase1 | / | / | 2 units (0.5-2 µl) | / | |
| PCR water | Variable to 20 µl | Variable to 20 µl | Variable to 20 µl | Variable to 20 µl | |
| Incubation time | 4 hours | Same time as test sample | Same time as test sample | 14 to 16 hours |
Incubation
- Incubate reaction tubes for the specified durations.
- After the incubation period, remove the tubes from the water bath and briefly place them on ice to stop any further the reaction.
Checking and interpreting results:
-
After the incubation period, prepare a 1.5% agarose gel, run and visualized as described in this protocol.
-
Supercoiled DNA should run faster (as would be expected in the negative control lane). Meanwhile, linear DNA should run true to size (known size of the plasmid used), and relaxed circle DNA should run slower in a single strand cut (as would be expected in the positive control lane).
-
Any change in the band intensity and migration position of the plasmid DNA band in the test sample lane compared to that of the negative control signals potential endonuclease activity of the test sample.
