Standard PCR with OnePCR™
1. For each 50 μLreaction,assemble the following components in a 0.2 mL PCR tube on ice just prior to use: 45 μL OnePCR™ (=1x), variable amount of forward and reverse primer (to final conc. of 0.1-0.2 μM), DNA template (final conc. 4pg-500 ng), total volume = 50 μL
2. Mix gently. If necessary, centrifuge briefly. Cap tubes and place in the thermal cycler.
Process in the thermal cycler for 25-35 cycles as follows: Initial Denaturation: 2-5 minutes at 94 °C, Denaturation: 20-40 seconds at 94 °C, Annealing: 1 min at the proper annealing temperature (30 cycles), Extension: 2 min at 72 °C, Final extension: 5 min at 72 °C
Note: Optimal conditions for amplification will vary depending on the primers and thermal cycler used. It may be necessary to optimize the system for individual primers, template and thermal cycler.
4. After the PCR reaction, RNA DNA electrophoresis will detect the PCR product.
5. Use the UV or blue-light transilluminator or UV epi-illuminator to photograph the gel.
Note: When the DNA concentration is less than 4pg, it may cause the migratory shift when performing the electrophoresis. To remedy this observation, we recommend to conduct the following steps ( please refer to the experimental procedures), or use the PCR Clean-Up & Gel Extraction Kit to remove the Novel Green prior to post-staining with the Novel Green again for restoring the DNA molecular weight in the original position.
Removel of Novel Green:
1. Immerse the PCR product containing the Novel Green into the 100 mM NaCl and add 2.5 volumes of absolute or 95 % ethanol.
2.Incubate on ice for 20 minutes.
3.Centrifuge the mixture at 4 °C for at least 10 minutes.
4. Remove the suspension of ethanol and wash the pellet with 1 mL of 70 % ethanol.
5. Dry the residual ethanol and resuspend the double-stranded DNA in the TE.