Specification for RNA Nucleic Acid Extraction and Purification Kit

Characteristic advantages:

1.Specificity: All primers used in the product have undergone detailed bioinformatics analysisGenBankBy comparing with a large self built database, it is ensured that each primer used is a species or serotype specific gene sequence segment, which can achieve specific detection of bacterial species and serotypes, with specificity reachingJia .

2. Reproducibility: All products in this series have been validated by a large number of experimental strains, with a reproducibility ofJia .

3. Sensitivity: This series of products can achieve highly sensitive detection of bacteria when the concentration of bacteria in the sample reaches103cfu/mlAt this time, direct detection of it can be achieved without the need for tedious bacterial growth processes.

4. Practicality: Wide detection range, covering more serious harm to human body17Planting respiratory and intestinal pathogenic bacteria can achieve rapid detection of clinical samples and other environmental samples. The entire detection process is3-4An hour.

5. advantage1Rich sequence resources, apart fromGenBankIn addition to the published sequences, the company has also conducted extensive sequencing of bacterial strains to theoretically ensure that the selected primers have good conservation and specificity.

6. advantage2This series of test kits has undergone extensive conservative and specific experimental verification. With the company's abundant bacterial resources, each detection kit has undergone extensive testing20Conservatism verification of more than standard strains and clinical strains40The specificity verification of more than two closely related standard strains and clinical strains ensures that there will be no false positive or false negative reporting results during use.

Extraction steps:

1. Take out the sample from the negative 80 freezer and slowly thaw it on ice; (Pre cool the centrifuge to 4 degrees in advance)

2. After thawing (red), add 200 μ l of chloroform (the volume of chloroform added is 1/5 of the volume of Trizol), shake vigorously (milky red), and let it stand on ice for 10 minutes. Chloroform is an organic solvent that effectively separates the organic and inorganic phases. In the organic phase, phenols and proteins mainly bind, causing proteins and RNA to detach and RNA to enter the aqueous phase. ）

3. Place it in a pre cooled centrifuge and centrifuge (4 degrees, 12000 rpm, 15 minutes). After centrifugation, it can be seen that there are three distinct layers (the upper layer is a colorless and transparent water phase for RNA layer, the middle layer is a very thin milky white layer for DNA layer, and the lower layer is a red layer for protein layer).

4. Carefully and slowly aspirate the supernatant, about 400 μ l (preferably less than more), and place it in another new 1.5 ml RNase free EP tube. Add an equal volume of isopropanol, invert and mix well, and let it stand at room temperature for 15 minutes.

5. Centrifuge (4 degrees, 12000 rpm, 15 minutes), white precipitate can be seen (sometimes there are few cells and no precipitate can be seen, it can be left for negative 20 or negative 82 hours before continuing with the following steps)

6. Discard the supernatant, add 950 ul of 75% ethanol to each tube, invert and mix well (remember not to use a gun to blow and mix well, as this will cause RNA dissolution).

7. Centrifuge (4 degrees, 12000 rpm, 10 minutes), visible white precipitate at the bottom.

8. After centrifugation, discard the supernatant and place it in the centrifuge for instant separation again. Use a 10 μ l pipette to wash away the residual liquid and open the lid to dry (about 5 minutes).

9. Add an appropriate amount of DEPC water to each sample according to the size of the precipitate (usually 20-30 μ l, sometimes no precipitate can be seen, 10 μ l DEPC water can be added for dissolution), blow and dissolve RNA, measure the concentration with an enzyme-linked immunosorbent assay (ELISA) reader on the eleventh floor, label with an OD value (260/280=1.8-2.0), and store at negative 80.

Note: For neutrophil extractionRNA suggests that the number of cells should be greater than 2x106 per sample;

remarkWear a mask during the operation.

Five elements of PCR reaction:
participateThere are five main substances involved in PCR reactions, namely primers, enzymes dNTP、 Template and Mg2+
Primers:Primers are crucial for PCR specific reactions, and the specificity of PCR products depends on the degree of complementarity between primers and template DNA. In theory, as long as any template DNA sequence is known, complementary oligonucleotide chains can be designed as primers, and PCR can be used to amplify the template DNA in large quantities in vitro.
The design of primers should follow the following principles:
① Primer length: 15-30bp, commonly around 20bp.
② Primer amplification span: 200-500bp is suitable, and under specific conditions, the fragment can be expanded to a length of 10kb.
③ Primer base: A G+C content of 40-60% is recommended. Insufficient G+C results in poor amplification, while excessive G+C can lead to non-specific bands. ATGC should be distributed randomly to avoid a string arrangement of more than 5 purine or pyrimidine nucleotides.
④ Avoid secondary structures within primers and avoid complementarity between two primers, especially at the 3 'end, as this can result in primer dimers and non-specific amplification bands.
⑤ The base pairs at the 3 'end of the primer, especially the last and last bases, should be strictly paired to avoid PCR failure caused by mismatched end bases.
⑥ Primers may have or can add suitable enzyme cleavage sites, and the amplified target sequence has suitable enzyme cleavage sites, which is beneficial for enzyme cleavage analysis or molecular cloning.
⑦ Primer specificity: Primers should have no apparent homology with other sequences in the nucleic acid sequence database.

Precautions:

1. The kit should be balanced at room temperature for 15-30 minutes before use when taken out from the cold storage environment. If the enzyme coated plate is not used up after opening, the Flat noodles should be stored in a sealed bag. Concentrated washing solution may precipitate crystals. When diluting, it can be dissolved by heating in a water bath, and washing does not affect the results.

2. Sample dispensers should be used for each step of sample addition, and their accuracy should be regularly checked to avoid experimental errors. The sample addition time should be controlled within 5 minutes. If there are a large number of specimens, it is recommended to use a sampling gun for sample addition.

3. Please make a standard curve at the same time as each measurement，Make a duplicate hole. If the content of the substance to be tested in the specimen is too high (sample)If the OD value is greater than the OD value of one standard well di, please dilute the sample diluent by a certain multiple (n times) before measuring. When calculating, pleaseagainMultiply by the total dilution factor（×n×5）。

4. The sealing film is only for one-time use to avoid cross contamination.

5. Please store the substrate away from light.

6. Strictly follow the instructions for operation, and the judgment of the test results must be based on the reading of the enzyme-linked immunosorbent assay reader

7. All samples, detergents, and various waste materials should be treated as infectious agents.

8. Components from different batch numbers of this reagent must not be mixed.

9. Expired products cannot be used.

10. If there is any discrepancy with the English manual, the English manual shall prevail.

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