Nemipterus Photosynthesis Lab Report
3.2 DNA Extraction
The DNA will be extracted from the Nemipterus samples according to Wizard® Genomic DNA Purification Kit (Promega) protocols. The first step of is cells and nuclei will be lysed by adding 120 µl of 0.5 Molar ethylenediaminetetraacetic acid (EDTA) to 500 µL of Nuclei Lysis Solution in a eppendorf tube, then it will be chilled on ice until the solution turn cloudy. The second step is 0.5 cm of Nemipterus sample tissue will be minced to fine powder. The fine powder of fresh Nemipterus tissue will be transfer into 1.5 ml eppendorf tube and labeled respectively according to species. A total of 600 µl of EDTA/Nuclei Lysis Solution will be pipetted from the mixture of first step and will be added to the tube containing Nemipterus tissue. The third step is, 17.5 µl of 20 mg/ml Proteinase K will be …show more content…
The mixture will be incubated for overnight at 50 °C with gentle shaking. Then the solution will let to cool down at room temperature for 5 minutes. Then 200 µl of Protein Precipitation Solution will be added to the solution and vortex at high speed for 20 seconds. The next step is, the mixture will be centrifuge for 4 minutes at 13,000-16,000 x g, white pellet supernatant will be formed in the tube and it will be transferred into 1.5 ml eppendorf tube which containing 600 µl of isopropanol at the room temperature. Then the mixture will be mixed by inverting the tube until white thread-like strands form as a visible mass. Then the solution will be centrifuged for 1 minute at 13,000-16,000 x g and the supernatant carefully decanted. After that, the DNA will be washed by adding 600 µl of 70 % ethanol into the tube and the solution will be mixed for few
The DNA will be extracted from the Nemipterus samples according to Wizard® Genomic DNA Purification Kit (Promega) protocols. The first step of is cells and nuclei will be lysed by adding 120 µl of 0.5 Molar ethylenediaminetetraacetic acid (EDTA) to 500 µL of Nuclei Lysis Solution in a eppendorf tube, then it will be chilled on ice until the solution turn cloudy. The second step is 0.5 cm of Nemipterus sample tissue will be minced to fine powder. The fine powder of fresh Nemipterus tissue will be transfer into 1.5 ml eppendorf tube and labeled respectively according to species. A total of 600 µl of EDTA/Nuclei Lysis Solution will be pipetted from the mixture of first step and will be added to the tube containing Nemipterus tissue. The third step is, 17.5 µl of 20 mg/ml Proteinase K will be …show more content…
The mixture will be incubated for overnight at 50 °C with gentle shaking. Then the solution will let to cool down at room temperature for 5 minutes. Then 200 µl of Protein Precipitation Solution will be added to the solution and vortex at high speed for 20 seconds. The next step is, the mixture will be centrifuge for 4 minutes at 13,000-16,000 x g, white pellet supernatant will be formed in the tube and it will be transferred into 1.5 ml eppendorf tube which containing 600 µl of isopropanol at the room temperature. Then the mixture will be mixed by inverting the tube until white thread-like strands form as a visible mass. Then the solution will be centrifuged for 1 minute at 13,000-16,000 x g and the supernatant carefully decanted. After that, the DNA will be washed by adding 600 µl of 70 % ethanol into the tube and the solution will be mixed for few