Our particle aims to extract blood DNA simply and conveniently and compare the DNA concentrations between new and old sample of necrophilic flies. We try investigating red blood DNA extraction case effectively extract DNA from fly, and is there a change between DNA concentrations from new and old sample of necrophilic flies? We provided a method for extracting fly DNA using ordinary blood test kit, which solved the problem of expensive and scarce insect extraction kit that pave the way for molecular biology analysis forming sets.
After solving the problem of extracting fly DNA, necrophilic files to either the time of death. To begin, sacrifice flies with ethyl acetate and place the samples in three gas vials. Weigh the samples and label them as fresh one, two, and three.
Next, retrieve 2-year-old samples stored in alcohol-filled centrifuge tubes. Absorb surface liquid with filter paper and weigh the samples. Also, retrieve 2-year-old samples stored in centrifuge tubes and weigh them.
Then, add a small amount of liquid nitrogen to a stainless steel container and wait until all liquid nitrogen has evaporated. Place a fly sample in the container and slowly pour liquid nitrogen. Freeze the samples in liquid nitrogen and remove them after the liquid nitrogen evaporates.
Separate the thorax individually, followed by slicing each thorax. Place the slices in a two milliliter centrifuge tube. To begin, place the clipped fly thorax samples in a two milliliter centrifuge tube.
Add 180 microliters of tissue lysis buffer to the tube, followed by 20 microliters of proteinase K.Vortex thoroughly at 3, 000 RPM for 10 seconds and incubate at 56 degrees Celsius until tissues are lysed. After incubation, vortex the samples again for 15 seconds. Add 200 microliters of lysis buffer and mix thoroughly by vortexing.
Then, add 200 microliters of ethanol and vortex again for 15 seconds. Next, place a DNA absorption column in a two milliliter collection tube and pipette the mixture into the column. Centrifuge at 6, 000 G for one minute.
Discard flow through in the collection tube. Place the column in a new tube and add 500 microliters of protein removal buffer. Centrifuge for one minute and discard the flow through.
Transfer the column to a new tube. Add 500 microliters of desalination buffer and centrifuge at 20, 000 G for three minutes to dry the membrane. Then, add 100 microliters of DNA elution buffer onto the membrane.
Incubate at room temperature for one minute and then centrifuge at 6, 000 G for one minute to elute the DNA. Store the DNA samples as appropriate. 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 photoanalysis. 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.
