DNA extraction and purification are often the first step in molecular biology experiments. It is the basis of various experiments such as gene cloning, molecular labeling, and hybridization detection. The quality of DNA is even directly related to the success or failure of subsequent experiments. Here we summarize the principles, methods, result analysis, difficult problems and other aspects of DNA extraction and purification. We hope to help you at the beginning of the experiment. We can use machines to assist our experiments-DNA extractor, RNA extractor to ensure Get better results.
Tools/Materials
Preparation: absolute ethanol, PBS, 15ml centrifuge tube
Method/Step
10.5mL of BIOG precipitate and 59.5mL of absolute ethanol
Add 42mL of absolute ethanol to 18mL of BIOG washing solution
Take a 15ml centrifuge tube, add 7.5mL of BIOG binding solution, then add 2.5mL of blood, gently shake to mix, leave at room temperature for 5 minutes, and centrifuge at 5,000 rpm for 3 minutes.
Discard the supernatant completely, add 625μL PBS, and suspend the pellet; add 2500μLBIOG lysate and 250μLBIOG digestion solution, shake and mix thoroughly, and bathe at 56°C for 10 minutes until the cells are completely lysed.
Add 6250μL of the prepared precipitation solution, gently invert and mix.
Put the adsorption column into the collection tube, transfer 4813ul of the above solution into the adsorption column, let it stand for 2 minutes, centrifuge at 5000 rpm for 2 minutes, and discard the waste liquid in the collection tube.
Put the adsorption column back into the collection tube, add 5000 μL of the prepared washing solution to the adsorption column, centrifuge at 5000 rpm for 1 minute, and discard the waste liquid in the collection tube.
Put the adsorption column back into the collection tube and centrifuge at 5000 rpm for 2 minutes to remove the remaining washing solution.
Take out the adsorption column, put it into a new 15 mL centrifuge tube, add 250–400 μLBIOG eluent, let it stand for 3 minutes, centrifuge at 5000 rpm for 2 minutes, and collect the DNA solution. The extracted DNA can be used in the next experiment or stored at -20°C.
Precautions
If precipitation occurs in the prepared liquid, it can be dissolved at 37°C and used after shaking.
If there is a translucent suspended substance in the fourth step, it will not affect the extraction of DNA and subsequent experiments
If you need to concentrate nucleic acids, you can proceed to the next steps. Add 800ul of absolute ethanol to the eluent, mix gently by inverting, centrifuge at 5,000 rpm for 5 minutes, discard the supernatant, and leave it at room temperature with the lid open for 5 minutes. After the ethanol evaporates, add 30–50ul of the eluent to dissolve the nucleic acid. The extracted DNA can be used in the next experiment or stored at -20℃
