3.2.4 Genomic DNA Extraction Of Onion Ixins
3.2.4 Genomic DNA extraction of the root tips
After the treatment of the onion bulbs with lake water sample at different period of time, the DNA from the root tips will be ready to be extracted and observed. First of all, the genomic of the Allium cepa will be easily done by the already available Uneasy® Plant Mini kit (Qiagen, Germany) in the laboratory. All of the procedures can be performed by referring to the manual provided by the kit. A very small amount of root tips that weigh 100 mg will be homogenized by using liquid nitrogen until the root tips turn to fine powder form. Next is the degradation of RNA step by adding 400 µl Buffer AP1 RNase to the sample in eppendorf tube. The mixture is incubated at 65°C for 10 minutes period after being vortex occasionally. In this period of time, the tube will be inverted 2-3 times during incubation. Then lysis step will take over by adding 130 µL Buffer P3. The solution then will be incubated for 5 minutes on ice until the tissue are completely lysed. The lysate solution will be centrifuged at 14,000 rpm for 5 minutes. The supernatant will be transferred carefully without disturbing the pellet to a sterile appendorf tube and 1.5 volumes (to the supernatant solution) of Buffer AW1 into the tube. The solution then will be mixed by pippetting …show more content…
The only difference of the PCR visualization process is the agarose concentration. In this step, the concentration of agarose that will be used is 1.5%. 1.5 g of agarose will be dissolved in 100 mL of 10X TBE buffer to obtain 1.5% of agarose concentration. Then, 100 bp DNA ladder will be used in this process because the amplicon has a smaller size compared to the genomic DNA. Only 2 µL of PCR product will be loaded together with the loading dye to get a medium intensity of the DNA band. The remaining procedures will be the same as the previous
After the treatment of the onion bulbs with lake water sample at different period of time, the DNA from the root tips will be ready to be extracted and observed. First of all, the genomic of the Allium cepa will be easily done by the already available Uneasy® Plant Mini kit (Qiagen, Germany) in the laboratory. All of the procedures can be performed by referring to the manual provided by the kit. A very small amount of root tips that weigh 100 mg will be homogenized by using liquid nitrogen until the root tips turn to fine powder form. Next is the degradation of RNA step by adding 400 µl Buffer AP1 RNase to the sample in eppendorf tube. The mixture is incubated at 65°C for 10 minutes period after being vortex occasionally. In this period of time, the tube will be inverted 2-3 times during incubation. Then lysis step will take over by adding 130 µL Buffer P3. The solution then will be incubated for 5 minutes on ice until the tissue are completely lysed. The lysate solution will be centrifuged at 14,000 rpm for 5 minutes. The supernatant will be transferred carefully without disturbing the pellet to a sterile appendorf tube and 1.5 volumes (to the supernatant solution) of Buffer AW1 into the tube. The solution then will be mixed by pippetting …show more content…
The only difference of the PCR visualization process is the agarose concentration. In this step, the concentration of agarose that will be used is 1.5%. 1.5 g of agarose will be dissolved in 100 mL of 10X TBE buffer to obtain 1.5% of agarose concentration. Then, 100 bp DNA ladder will be used in this process because the amplicon has a smaller size compared to the genomic DNA. Only 2 µL of PCR product will be loaded together with the loading dye to get a medium intensity of the DNA band. The remaining procedures will be the same as the previous