Collect appropriate biological samples, such as blood, tissues, cells, saliva, etc., according to the requirements of the kit. Ensure that the sample collection and preservation comply with the specifications to avoid sample degradation or contamination.
For tissue samples, operations such as grinding and homogenization may be required to fully break the cells and release nucleic acids. For cell samples, the cells need to be collected first and then washed.
Cell Lysis
Add the prepared sample to a centrifuge tube containing lysis buffer. Mix thoroughly to make the cells rupture under the action of the lysis buffer, releasing the nucleic acids inside. The lysis time and temperature may vary depending on the kit and sample type. Generally, incubation is required at room temperature or a specific temperature for several minutes to tens of minutes.
Nucleic Acid Binding
Add binding buffer and other reagents to the lysed sample to make the nucleic acids bind to a specific solid – phase carrier (such as silica membrane, magnetic beads, etc.). Usually, operations such as vortexing and inverting for mixing are required to ensure that the nucleic acids and the carrier are in full contact and bind.
For kits using magnetic beads, place the centrifuge tube on a magnetic stand at this time so that the magnetic beads adsorb to the tube wall, and then remove the supernatant. For kits using silica – membrane spin columns, transfer the mixture to the spin column, and centrifuge to make the nucleic acids bind to the silica membrane. Then pour out the waste liquid in the collection tube.
Washing of Nucleic Acids
Add wash buffer to wash the nucleic acids bound to the carrier to remove impurities and residual proteins and other contaminants. Multiple washes are generally required, and the wash buffer should be removed each time.
The centrifugation conditions and time during the washing process also need to be operated according to the kit instructions to ensure thorough washing without affecting the binding of nucleic acids.
Nucleic Acid Elution
Add an appropriate amount of elution buffer, such as nuclease – free water or TE buffer, to the carrier to make the nucleic acids detach from the carrier. Collect the eluate, which contains the extracted nucleic acids.
During elution, incubation at a certain temperature for several minutes may be required to improve the elution efficiency. Then collect the eluate into a new centrifuge tube by centrifugation or magnetic adsorption.
Precautions
Reagent Storage and Use
Store various reagents in the kit according to the temperature and conditions specified in the instructions to avoid reagent failure. Balance the reagents to room temperature before use to prevent phenomena such as precipitation due to temperature differences.
Pay attention to the expiration date of the reagents. Check whether the reagents have abnormal conditions such as color change and turbidity before use. If so, do not use them.
Use sterile pipettes and tips when taking reagents to avoid reagent contamination. Do not mix the tips for different reagents to prevent cross – contamination.
Operation Environment and Equipment
The operation should be carried out in a clean and sterile environment, preferably in a biological safety cabinet or laminar – flow hood to reduce external nucleic acid contamination.
Ensure that the centrifuge tubes, pipette tips, grinding equipment, etc., used are free of nuclease contamination. Equipment that has been autoclaved or treated to be nuclease – free can be used.
Regularly calibrate and maintain instruments such as pipettes to ensure their accuracy and stability, and to ensure the accuracy of the sample volume added.
Sample Handling
Strictly follow the operating procedures during sample collection to ensure the quality and representativeness of the sample. Avoid using too little or too much sample, which may affect the nucleic acid extraction effect.
When handling different samples, pay attention to changing gloves and equipment to prevent cross – contamination between samples. For infectious samples, operate in accordance with the relevant biosecurity regulations and take protective measures.
Operation Steps and Time Control
Strictly follow the operation steps and time requirements in the kit instructions. Do not change the operation sequence or extend/shorten the operation time at will, so as not to affect the extraction efficiency and quality of nucleic acids.
When performing operations such as centrifugation, ensure that the centrifuge tubes are placed evenly to avoid damage to the instrument or loss of samples due to unbalanced centrifugation.
Result Detection and Storage
The extracted nucleic acids should be detected or analyzed as soon as possible. If not used in a timely manner, store the nucleic acids at an appropriate temperature. Generally, DNA can be stored at – 20°C, and RNA needs to be stored at – 80°C. Avoid repeated freezing and thawing to prevent nucleic acid degradation.
Before nucleic acid detection, methods such as agarose gel electrophoresis and ultraviolet spectrophotometry can be used to detect the quality and concentration of the extracted nucleic acids to ensure that the quality of the nucleic acids meets the requirements of subsequent experiments.
HUACHENGYANG
