To begin, extract DNA from fly samples using a DNA extraction kit. Then use a microplate reader to determine the values of OD260 and OD280 to compare DNA purity and nucleic acid concentration. Next, prepare fluorometer standard solutions by adding 190 microliters of working solution to two 0.5 milliliters centrifuge tubes.
Then add 10 microliters each of standard one and standard two to the working solutions. Keep the tubes away from light for at least 15 minutes. Mix two microliters of test DNA with 198 microliters of working solution in a 0.5 milliliter centrifuge tube and keep the mixture in the dark for at least 15 minutes.
After 15 minutes, turn on the fluorometer and select the dsDNA concentration measurement, long range setting. Test standard one and standard two to obtain the standard curve. Then place the samples to be tested in the assay wells.
Adjust the DNA spiking volume to two microliters and perform the measurement. To visualize the DNA first set up 20 microliters of the PCR amplification reaction. Place the tubes in a thermal cycler and run the PCR cycles.
After PCR, subject the products to 2%agarose gel electrophoresis. Then place the gel on a gel imaging system for photo analysis. Assessment of extracted DNA purity by Microplate reader showed that all fresh samples and old sample one had OD260 to OD280 ratios above two.
In contrast, old sample two displayed a lower ratio. DNA concentration derived from OD260 readings was highest in fresh samples, followed by old sample one and lowest in old sample two. Electrophoretic analysis revealed bands in the range of 250 to 400 base pairs, which were more pronounced in fresh samples, compared to old ones.
