Nucleic acid molecular chain is generally long, the use of ordinary drop or spray method is easy to make its structure damaged. Therefore, at present, most of the protein single molecule membrane technology for the preparation of nucleic acid molecular samples. Available unfolding method, diffusion method, one-step dilution method to make nucleic acid adsorption to the protein single molecule membrane. This experiment uses the unfolding method. Content from the microbiology laboratory (third edition)
Operation method
Nucleic acid samples
Principle
Many globular proteins can form an insoluble denaturing film on the surface of aqueous or saline solutions, and under appropriate conditions this film can become a monomolecular layer, consisting of a molecular network of extended peptide chains. When the nucleic acid molecules and the protein monomolecular membrane will be due to the role of the protein amino acid basic side chain groups, so that the nucleic acid from the three-dimensional spatial structure of the solution configuration adsorbed on the peptide chain network into a two-dimensional spatial configuration, and from the morphology to the structure of a certain degree of integrity can be maintained. Finally, the protein single-molecule membrane with adsorbed nucleic acid molecules was transferred to the carrier membrane, and the contrast of the sample was increased by negative staining and other methods for electron microscopic observation.
Materials and Instruments
Plasmid pBR322 Move 1. Mix plasmid pBR322 with an alkaline globular protein solution (usually cytochrome c). Bring the concentrations to 0.5-2 mg/ml and 0.1 mg/ml, respectively, and add ferric acetate and disodium EDTA at a final concentration of 0.5-1 mol/L and 1 mmol/L, respectively, to make the unfolding solution, pH 7.5. 2. 2. Fill a clean dish with a certain lower phase solution (distilled water or 0.1-0.5 mol/L ferric acetate solution) and add a small amount of talcum powder on the liquid surface. Place a clean slide diagonally in the dish, use a microsyringe or pipette gun to suck 50 μl of the unfolding solution, swing back and forth on the slide about 1 cm away from the surface of the lower-phase solution, drop on the surface of the slide, then you can see the talcum powder layer recedes, indicating that the protein single-molecule membrane is gradually formed, and the whole process will take about 2-3 min. 3. 3. After the formation of single molecular membrane, with the electron microscope silver take a carrier network covered with a support film, so that the support film facing down, placed in the single molecule membrane from the leading edge of the membrane 1 cm or 0.5 cm from the surface of the membrane of the slide, and immediately fished up with tweezers. The single molecule membrane is adsorbed on the supporting membrane, and the excess liquid can be absorbed by a small piece of filter paper, or the carrier mesh can be floated directly in anhydrous ethanol for 10~30 s. 4. 4. The carrier mesh containing the monomolecular membrane is stained in a 10-3 to 10-5 mol/L ethanol solution of uranyl acetate for about 30 s (this step can be carried out at the same time as dehydrating with ethanol), or the metal is spray-plated on the surface of the sample of nucleic acid by the method of rotary projection. It is also possible to combine the two methods and perform the projection after staining, which is sometimes more effective than using one method alone. Caveat 1. When the single molecule membrane is formed, the whole device should be covered with a glass cover to prevent the influence of airflow caused by the operator's breathing and bystanders' movement and the contamination of dust and other dirty things. 2. in the unfolding solution can be added to the appropriate amount of nucleic acid and the amount of difference between the indication of the specimen, such as tobacco mosaic disease virus, etc., in order to facilitate the identification of the unfolding of monomolecular membranes and the back of the transfer of the good and bad. 3. When the protein forms a single molecule membrane, the nucleic acid molecules in solution are also distributed in the middle of the protein base membrane at the same time, and slightly wrapped by the protein peptide chain. Theoretical calculations and experiments have proved that when 1 mg of protein is unfolded into a good monomolecular membrane, its area is about 1 m2When 1 mg of protein unfolds into a good monolayer, the area is about 1 m 2 . Thus, the degree of goodness can be estimated based on the size of the area of the final monomolecular membrane. If the area is too small. It means that the membrane formed is not a monomolecular layer, and thus there is a risk that the nucleic acid will be partially or completely encapsulated by the membrane, so that the entire nucleic acid molecule disappears or the contrast is bad. 4. The angle of tilt of the slide determines the speed of the unfolding liquid sliding down to the next phase of the solution, and has an impact on the quality of the formation of single molecule membrane, experience has proved that the tilt to 15 ° or so is appropriate. For more product details, please visit Aladdin Scientific website.
Basic globular protein solution Iron acetate Disodium ethylenediaminetetraacetic acid Distilled water Talcum powder Anhydrous ethanol Uranium acetate ethanol solution
Microsyringe Pipette gun Slide Flat dish Carrier mesh with supporting membrane Tweezers Filter paper