Tris base is a common buffer component commonly used in biochemical and molecular biology experiments. It has functions such as stabilizing pH value and providing an ionic environment. During the DNA extraction process, trimethylaminomethane is often used as an important component of the extraction buffer to ensure that the extracted DNA has high quality and yield. The following will provide a detailed introduction to the method and steps of DNA extraction using trimethylaminomethane.
Tris, NaOH, NaCl, ethanol, DNA samples (such as blood and tissue), water, centrifuge tubes, centrifuges, pipettes, and other experimental equipment.
1. Sample preparation: Crush and homogenize the DNA sample to be extracted for cell lysis. Depending on the type of sample, different crushing methods can be used, such as mechanical crushing, ultrasonic crushing, etc. After crushing, homogenize the sample to fully disperse the cell fragments in the buffer solution.
2. Buffer preparation: according to the experimental requirements, add trimethylolaminomethane, NaCl and ethanol into deionized water in a certain proportion, and adjust the pH value to a suitable range (usually 7.5-8.5). Add an appropriate amount of NaOH to raise the pH value of the solution to a certain range for subsequent cell lysis steps.
3. Cell lysis: Add the fragmented sample to an appropriate amount of trimethylaminomethane buffer to fully dissolve the sample. Add an appropriate amount of glass beads, gently shake the centrifuge tube until the glass beads come into complete contact with the sample, crush the cells, and then place the mixture in a centrifuge for centrifugation at an appropriate speed. The purpose of this step is to separate the cell fragments from the glass beads, which remain in the sediment while the glass beads float on top of the solution.
4. Remove glass beads: Pour out the upper layer of solution, dissolve the precipitate again in an appropriate amount of trimethylaminomethane buffer, and remove unbroken cells and residual glass beads. This step can be achieved by centrifugation or filtration again.
5. DNA precipitation: Add an appropriate amount of ethanol to the dissolved sample to precipitate DNA, with the aim of removing other impurities from the solution. Control the amount and precipitation time of ethanol to ensure complete DNA precipitation without loss.
6. Centrifuge separation: Centrifuge the mixture, collect the precipitated DNA, place the centrifuge tube in a centrifuge, and centrifuge at an appropriate speed. Collect DNA precipitates from the supernatant and dissolve them in an appropriate amount of trimethylaminomethane buffer.
7. Purification and washing: According to experimental requirements, suitable purification methods can be selected to further remove impurities and enhance the purity of DNA. Common purification methods include magnetic bead method, etc. After purification, perform a washing step to remove impurities and salt ions remaining in the DNA sample.
8. Quality testing: Conduct quality testing on the extracted DNA, such as UV absorption testing, electrophoresis testing, etc., to ensure that the extracted DNA has high quality. In addition, PCR amplification and other experiments can be conducted to verify whether the extracted DNA can be used for subsequent experiments and analysis.
9. Storage and transportation: Store the extracted DNA under suitable temperature and humidity conditions, and ensure that it is not affected by external factors during transportation. It is recommended to use professional storage equipment or have professionals handle and transport DNA samples to ensure their stability and safety.
Matters needing attention
1. Using high-quality experimental materials and reagents: Select high-quality experimental materials and reagents, such as high-quality DNA extraction reagents, high-purity trimethylaminomethane, etc., to ensure that the extraction process is not disturbed.
2. During the entire operation process, it is necessary to avoid DNA contamination and degradation, such as using aseptic techniques and accurately adjusting the pH value of the trimethylaminomethane buffer.
3. Strictly control the experimental conditions, such as temperature, time, ion concentration, etc., to achieve the best extraction effect and quality.
4. Optimization of extraction steps: Optimizing the extraction steps, such as adjusting washing times, optimizing precipitation steps, etc., can improve the yield and purity of DNA.
Desheng is a supplier of biological buffering agents, which can produce raw material powders with a purity of up to 99% for reagent manufacturers to prepare and use. The raw materials undergo strict quality control to ensure their stability, safety, and effectiveness. In addition, we also provide professional technical support to help customers solve problems during use one-on-one, ensuring the smooth progress of experiments. If you have any relevant intentions, please feel free to contact us for purchase at any time!