What is the classic method of DNA extraction?
What is nucleic acid?Nucleic acid is a large biomolecule usually located in cells and is mainly responsible for carrying and transmitting the genetic information of an organism. There are two types of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Nucleic acid includes two kinds of molecules, DNA and RNA, which exist in the state of binding with proteins in cells. The main steps of nucleic acid extraction are:
* Lyse cells
Remove the proteins bound to nucleic acid and biological macromolecules such as polysaccharides and lipids, and remove other unwanted nucleic acid molecules. For example, when extracting DNA molecules, RNA should be removed, and vice versa.
* Precipitate nucleic acid
Purify nucleic acid, remove impurities such as salts and organic agents
What is the classic method of DNA extraction?
The classic method of DNA extraction, the so-called phenol-chloroform extraction method. Because it is easier to remove proteins by alternately extracting two different organic solvents, the extraction sequence is phenol, phenol/chloroform (1:1), and chloroform. The DNA extracted by this method has high purity, large fragments and good effect. The disadvantage is that more complicated.
Illustrate:
When recovering the upper aqueous phase, be sure not to touch the interface between the two phases to avoid sucking proteins and other substances into the new centrifuge tube.
For precipitation of DNA, absolute ethanol is the organic solvent of choice. Another: isopropanol, sodium acetate, etc.
The purpose of rinsing DNA with 70% ethanol is to remove residual salts and impurities such as excess SDS and phenol, because SDS remains dissolved in 70% ethanol and does not co-precipitate with DNA, so this is removed by discarding the supernatant. Detergent to avoid influence on subsequent PCR reactions.
TE buffer: 10mmol/L Tris-HCl
1mmol/L EDTA pH 8.5 or 8
How to extract RNA?
RNA extraction conditions are stricter than DNA, mainly because there are a large number of RNases that strongly degrade RNA in clinical specimens and laboratory environments. RNase is a class of enzymes with very stable biological activity. This enzyme is resistant to acid, alkali and high temperature, and it cannot be completely inactivated by boiling.
Protein denaturing agents can temporarily inactivate it, but after the denaturing agent is removed, the activity can be restored. In addition to intracellular RNase, RNase exists in dust in the environment, various experimental utensils and reagents, human sweat and saliva. Therefore, when extracting RNA, it is important to avoid RNase contamination of the specimen and prevent RNase from degrading the extracted RNA.
Handling of Vessels and Preparation of Solutions for RNA Extraction
Plastic products: such as test tubes, EP tubes, and Tip, use sterilized disposables.
Glass supplies: such as beakers, test tubes and other supplies, which are often contaminated by RNase, must be dried at 180°C for more than 8 hours before use, or soaked in 0.1°C water (diethyl pyrocarbonate).
DEPC is a strong inhibitor of RNase, and its mechanism of action is to denature proteins by binding to histidine in proteins. Soak at 37°C for 2 hours, then rinse with sterilized water for several times, and dry bake at 100°C for 15 minutes to remove residual DEPC on the utensils.
Preparation of the required solution: The solution must be prepared with autoclaved water and special chemical reagents for RNA extraction, weigh the reagents with a dry-baked spatula, and place the solution into RNase-free glassware.
Strictly speaking, all solutions should be treated with 0.1 °C at 37°C for more than 12 hours, then heated at 100°C for 15 minutes or autoclaved for 15 minutes to remove residual DEPC.
Control of RNase Contamination in RNA Extraction
The main potential source of contamination is the operator's hands. There is no doubt that RNase will be left where the hands directly touch. In addition, the saliva brought out by speaking is also rich in RNase, so it should be worn once during all operations involving RNA. Sexual gloves and masks.
In experiments, gloves may contaminate RNase after touching "dirty" glassware and other objects, so gloves should be changed frequently during RNA extraction experiments.
Nucleic acid extraction supplier:
Geneture medical specialized in the field of nucleic acid extraction system, PCR detection system and laboratory consumables, main products involved in：
1.Auto nucleic Acid Extractor （32T, 96T）
2.PCR fluorescence quantitative analyzer (16T, 96T)
3.Nucleic Acid Extraction Kit (Magnetic beads method)
4.Lab consumables: deep well plate, mag-rod sleeve, pipette tips, VTM, swabs, PCR tubes, PCR plate, centrifuge tubes, cryogenic Vials, saliva collection, cell culture dish/flask/plate/tube and so on.
Please don’t hesitate to contact with us to get a quotation.