The commonly used method for testing the DNA composition of genetically modified soybeans using the Shanghai Luxiangyi centrifuge.

Release time:

2013-06-24

Overview:

Experimental requirements include commonly used laboratory instruments and equipment: Luxiangyi high-speed refrigerated centrifuge (12,000 rpm), Luxiangyi high-speed benchtop centrifuge (12,000 rpm), UV spectrophotometer, magnetic stirrer, and more.

Experimental requirements include commonly used laboratory instruments and equipment, such as the Luxiangyi high-speed refrigerated centrifuge (12,000 rpm), the Luxiangyi high-speed benchtop centrifuge (12,000 rpm), a UV spectrophotometer, a magnetic stirrer, an autoclave, a PCR amplification instrument, an electrophoresis apparatus, a UV transilluminator, a gel imaging system or photographic system, and a double-distilled water purification unit.

DNA Extraction from Samples—CTAB Method: Take 100 mg of thoroughly ground soybean sample, grind it completely into a fine powder in liquid nitrogen, and then add it to 400 μL of ice-cold CTAB extraction buffer I. Add 500 μL of CTAB extraction buffer II, preheated to 65°C, and gently invert the mixture occasionally to ensure thorough mixing. After allowing the mixture to cool down to room temperature, add the stock solution and incubate at room temperature for 30 minutes. Next, add 450 μL of chloroform-isopentanol, and gently invert the mixture to mix thoroughly.

Transfer the supernatant into a clean centrifuge tube, then sequentially add 600 μL of isopropanol and 60 μL of sodium acetate solution. Gently invert the tube several times to mix thoroughly. Centrifuge at 12,000 rpm for 10 minutes. Discard the supernatant, then add 100 μL of 70% ethanol to wash the precipitate. Centrifuge at 12,000 rpm for 5 minutes, and discard the supernatant again. After removing any residual ethanol, once the precipitate has dried, dissolve the DNA pellet in 100 μL of TE buffer and store it at -20℃ for future use.

DNA can also be extracted from samples using commercially available genomic DNA extraction kits. To determine and preserve the purity of the DNA solution, appropriately dilute the DNA, measure and record its absorbance at 260 nm and 280 nm, and calculate the concentration of the purified DNA based on the assumption that an OD260 value corresponds to a DNA concentration of 50 μg/mL. The DNA solution should have an OD260/OD280 ratio within the range of 1.7 to 2.0. Based on the measured concentration, dilute the DNA solution to a final concentration of 25–50 ng/mL and store it at -20°C. Note: Since genomic DNA is not suitable for repeated freeze-thaw cycles, it is recommended to aliquot frequently used DNA into multiple tubes for storage. When needed, take out the required amount, thaw it immediately, and use it right away. After use, any remaining DNA should be stored briefly at 4°C in a refrigerator, with a storage period not exceeding 14 days.

Common Troubleshooting for Laboratory Freezer Centrifuges That Do Not Display Data and Fail to Operate Normally

Precautions for using a refrigerated centrifuge in the pre-cooled state