Magnetic Pathogen DNA/RNA Kit HYD522
Magnetic Pathogen DNA/RNA Kit The magnetic bead-based pathogen DNA/RNA extraction kit is suitable for extracting pathogenic microorganism nucleic acids from biological fluid samples such as cervical swab protective fluid (it is recommended not to use protective fluid containing high concentrations of guanidine salts), whole blood, plasma, sputum, alveolar lavage fluid, cerebrospinal fluid and other samples. This method uses magnetic bead purification technology, and no toxic phenol chloroform extraction is required during the extraction process. It is safe, non-toxic and fast. This method uses a lysis method that combines chemical and mechanical methods. The purified nucleic acids include nucleic acids of microorganisms such as viruses, mycoplasmas, bacteria, and fungi. The obtained products can be directly used in PCR, qPCR, metagenomic library construction, DNA/RNA co-construction library and other experiments. Used in conjunction with magnetic bead-based automated extraction instruments, high-throughput extraction of nucleic acids can be achieved.
Product information
|
Components |
24T |
48T |
|
Proteinase K |
0.7 mL |
1.1 mL |
|
Lysis Enhancement Solution |
4 mL |
5 mL |
|
Grinding tube |
0.4 g×24 |
0.4 g×48 |
|
Lysis and Binding Solution |
20 mL |
33 mL |
|
Magnetic bead suspension |
0.7 mL |
1.1 mL |
|
Wash Buffer A |
20 mL |
40 mL |
|
Wash Buffer B |
40 mL |
80 mL |
|
Elution Solution |
4 mL |
6 mL |
Storage Conditions
Proteinase K should be stored at 2~8℃ and the other ingredients should be stored at room temperature. The shelf life is 18 months.
Sample Description
1. Applicable specimen types: cervical swabs, whole blood, plasma, sputum, alveolar lavage fluid, cerebrospinal fluid and other samples.
2. Specimen storage and transportation: The specimen can be used for testing immediately or stored at -70℃ or lower for testing. The storage period is 6 months, and repeated freezing and thawing should be avoided. The specimen is transported by cold chain.
3. Freezing and thawing requirements: quick freezing and thawing, avoid repeated freezing and thawing.
4. Swab-type protective fluid may contain high concentrations of guanidine salts, which will react with Lysis Enhancement Solution and result in poor extraction effect; if such samples need to be extracted, there is no need to add Lysis Enhancement Solution. After step 1 is processed, continue with step 4.
5. Whole blood samples: For whole blood with high cell content such as umbilical cord blood and bone marrow blood, magnetic beads or color residues may appear during the extraction process. It is recommended to dilute the sample with saline or PBS before nucleic acid extraction.
6. Sputum samples: Magnetic beads are likely to remain during the extraction process. It is recommended to dilute the sample with saline or PBS before nucleic acid extraction.
Operation
1. Sample processing
1.1 Select the corresponding steps for processing according to different sample types
A. Virus, mycoplasma, and chlamydia nucleic acid extraction: Take 200~300 μL liquid sample and 20 μL Proteinase K into a centrifuge tube, add 600 μL Lysis and Binding Solution, and extract according to step 4 without pre-treatment.
B. Non-fungal and easily lysable microbial nucleic acid extraction: Take 350~400 μL liquid sample into a Grinding tube, add 40 μL Lysis Enhancement Solution and 20 μL Proteinase K, and vortex at high speed for 1 min to mix.
Note: Easily lysable bacteria: Gram-negative bacteria such as Escherichia coli, Haemophilus influenzae, Legionella pneumophila, Bordetella pertussis, Vibrio cholerae, etc.
C. Extraction of nucleic acid from fungi and difficult-to-lyse microorganisms: Take 350~400 μL of liquid sample into a Grinding tube, add 40 μL of Lysis Enhancement Solution and 20 μL of Proteinase K, vortex at the maximum speed of the vortexer or transfer to a high-speed oscillator to grind the sample for 10 min (for different brands of oscillators, please choose the instrument recommended program).
Note: Fungi: Candida albicans, Candida tropicalis, Aspergillus flavus, Penicillium. Difficult-to-lyse bacteria: Gram-positive bacteria such as Staphylococcus, mold, Streptococcus pneumoniae, etc.
1.2 Transfer to a water bath or dry thermostat, warm at 70℃ for 20 min to further lyse the sample, take out and vortex to mix for 30 s after lysis.
1.3 If the solution is turbid after lysis, centrifuge at 10,000×g for 1 min; if the solution is clear and transparent after lysis, centrifuge for 30 s.
Note: If the supernatant of fungi such as Trichoderma is colored after centrifugation, you can contact the internal preparation of SPS precipitation solution and follow the following process: take 300 μL of the supernatant after centrifugation into a new centrifuge tube, add 100 μL of SPS precipitation solution and mix well, place on ice at 4℃ for 10 min, and centrifuge at 10,000×g for 3 min.
2. Single-tube operation
2.1 Pipette 200~300 μL of supernatant solution from step 1.3 into a new centrifuge tube, add 600 μL of Lysis and Binding Solution, and carefully pipette to avoid sucking up precipitates or glass beads.
2.2 Add 20 μL of Magnetic bead suspension, vortex mix for 30 s, and oscillate or vortex for 5~7 min to allow the magnetic beads to adsorb nucleic acids.
2.3 Transfer to a magnetic stand for magnetic separation until the solution is clear, and discard the solution.
2.4 Add 700 μL of Wash Buffer A, vortex for 30 s, transfer to a magnetic stand for adsorption until the solution is clear, and discard the solution.
2.5 Add 700 μL of Wash Buffer B, vortex for 30 s to break up the magnetic beads, and then transfer to a magnetic stand for magnetic separation until the solution is clear, and discard the solution.
2.6 Add 700 μL of Wash Buffer B again, vortex for 30 s to break up the magnetic beads, transfer to a magnetic stand for magnetic separation until the solution is clear, and discard the solution.
2.7 Centrifuge briefly to collect the droplets on the tube wall, transfer to a magnetic stand for clarification, and aspirate the residual liquid. Air dry for 3-5 min.
2.8 Add 30~100 μL of Elution Solution, vortex at high speed for 2~3 min to break up the magnetic beads. Incubate at 60ºC for 5 min. If there is no oscillating mixer, vortex 2~3 times during this period.
Note: Ethanol residue will inhibit subsequent enzyme reactions, so make sure that the ethanol evaporates completely when drying. Do not dry for too long to avoid affecting the subsequent elution effect.
2.9 Instantly centrifuge to collect the droplets on the tube cap and transfer to a magnetic stand until the magnetic beads are completely adsorbed, then carefully transfer the liquid to a new centrifuge tube to obtain the nucleic acid solution.
2.10 Nucleic acid solutions can be stored at -20°C for short term storage and -80°C for long term storage.
Notes
1. The various buffers in this kit contain guanidine salts. For your safety and health, please wear a lab coat and disposable gloves. And handle according to safety standard precautions. Do not let the buffer come into contact with the skin, eyes and mucous membranes. If it does happen, please wash immediately with plenty of water and seek medical attention.
2. If the solution precipitates, it needs to be bathed in 30℃ water until the precipitate is completely dissolved before use.
3. If the lysis enhancement solution precipitates, it needs to be bathed in 60℃ water until the precipitate is completely dissolved before use.
4. If the magnetic bead suspension is frozen, do not use it.
5. The various buffers in this kit contain guanidine salts. Do not use oxidizing disinfectants such as sodium hypochlorite to treat them, otherwise toxic gases will be released and they must be treated as medical waste.
6. There may be residual magnetic beads during elution. Try to avoid inhaling magnetic beads when aspirating samples.