| Description | High-Density Lipoprotein (HDL), as an anti-atherogenic lipoprotein, transports cholesterol from peripheral tissues to the liver for metabolism, where it is converted into bile acids or directly excreted from the intestine via bile. This process reduces cholesterol deposition on the arterial wall. High-Density Lipoprotein (HDL), as an anti-atherogenic lipoprotein, transports cholesterol from peripheral tissues to the liver for metabolism, where it is converted into bile acids or directly excreted from the intestine via bile. This process reduces cholesterol deposition on the arterial wall. HDL exerts its anti-atherosclerotic effects through various mechanisms, including promoting reverse cholesterol transport, anti-inflammatory and antioxidant activities, inhibiting thrombus formation, and improving endothelial cell function.Detection Principle: Cholesterol esterase (CHER) and cholesterol oxidase (CHOD) are chemically modified and used in conjunction with dextran sulfate and magnesium ions (or other compounds like sulfated cyclodextrin complexes) to reduce their enzymatic reactivity towards LDL, VLDL, and chylomicrons, making them selectively interact with HDL-cholesterol. Based on this principle, in the first reaction step, LDL, VLDL, and chylomicrons are complexed with reagents like dextran sulfate. In the second reaction step, using the chemically modified CHER and CHOD, HDL-cholesterol is directly measured without the need to separate other lipoproteins. Specifically, the chemically modified CHER catalyzes the hydrolysis of cholesterol esters to generate Free Cholesterol (FC). FC is then oxidized by CHOD to produce 4-cholestenone and hydrogen peroxide. Subsequently, hydrogen peroxide reacts with 4-aminoantipyrine and phenol under the catalysis of peroxidase (POD) to generate a red quinoneimine compound, which has a characteristic absorption peak at 546 nm. The HDL-C content is determined by measuring the absorbance at 546 nm.Component96TStorageReagent 118 mL2-8℃. Store in the dark.Reagent 26 mL2-8℃. Store in the dark.Reagent 31EA2-8℃. Store in the dark.Standard (Powder, 1 vial) Preparation:1. Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom of the tube.2. Add 0.1 mL of distilled water to dissolve. Use within one week. The prepared concentration is as indicated on the label.User-Prepared Instruments and Reagents:Mortar (Homogenizer), balance, ice box (ice maker), benchtop centrifuge, adjustable micropipettes, water bath (oven, incubator, metal bath), 96-well plate, centrifuge tubes, microplate reader, distilled water (deionized water or ultrapure water are acceptable), ethanol.Experimental ProcedureIt is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure and to determine or adjust sample concentrations based on the preliminary results, preventing unnecessary waste of samples or reagents.1. Sample Extraction1.1 Tissue SamplesWeigh approximately 0.1 g of tissue sample and place it in a mortar. Add 1 mL of ethanol and homogenize in an ice bath. Centrifuge at 12,000 rpm, 4°C or room temperature for 10 minutes. Collect the supernatant for assay.Note: If increasing the sample amount, maintain a tissue mass (g) to ethanol volume (mL) ratio between 1:5 and 1:10.1.2 Liquid SamplesAssay clear liquid samples directly. If turbid, centrifuge and use the supernatant for assay.1.3 Serum SamplesFor routine, clear serum samples, add reagents directly according to the assay table and proceed with detection. If the serum sample has a high protein content, adding reagents as per the table may cause turbidity. In this case, first take 200 µL of serum + 200 µL of ethanol, mix well by inverting several times, centrifuge at 8,000 rpm, 4°C or room temperature for 5 minutes, and then collect the supernatant for assay.1.4 Bacterial/Cell SamplesCollect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Add 1 mL of ethanol per approximately 5 million bacteria/cells. Disrupt the bacteria or cells by sonication in an ice bath (power 200W, pulse 3s on, 10s off, repeat 30 times). Centrifuge at 12,000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.*Note: If increasing the sample amount, maintain a bacteria/cell count (10⁴) to ethanol volume (mL) ratio between 500:1 and 1000:1.*2. Assay Steps2.1 Preheat the microplate reader for 30 minutes (or wait for the instrument to complete its self-check). Set the wavelength to 546 nm.2.2 Thaw all reagents to room temperature (25°C). Add reagents sequentially to a 96-well plate as follows:Reagent (µL)Test TubeStandard Tube (once)Blank Tube (once)Sample2.5Standard2.5Distilled Water2.5Reagent 1180180180Mix well and incubate at 37°C for 5 minutes. Read the absorbance at 546 nm for each tube (A₁).Reagent 2606060Mix well and incubate at 37°C for 10 minutes. Read the absorbance at 546 nm for each tube (A₂). Calculate ΔA = A₂ - A₁ for each tube.Note:(1) If the A₂ value for the Test Tube is greater than 1, dilute the sample with ethanol. The dilution factor (D) must be substituted into the calculation formula.(2) If ΔA for the Test Tube is lower than ΔA for the Blank Tube, consider increasing the sample volume V₁ (e.g., increase the sample volume in the Test Tube and the water volume in the Blank Tube to 5 µL or more, keeping Reagents 1 and 2 volumes unchanged; for the Standard Tube, keep at 2.5 µL and add 2.5 µL distilled water to make up volume) or increasing the sample weight W (e.g., to 0.2 g or more). The changed V₁ or W must then be substituted into the calculation formula.3. Calculation of Results3.1 Based on Sample MassDerived Formula:HDL-C (µmol/g weight) = (CStandard × V₂) × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ (W × V₁ ÷ V) × DSimplified Formula:HDL-C (µmol/g weight) = CStandard × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ W × D3.2 Based on Protein ContentDerived Formula:HDL-C (µmol/mg prot) = (CStandard × V₂) × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ (Cpr × V₁ ÷ V) × DSimplified Formula:HDL-C (µmol/mg prot) = CStandard × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ Cpr × D3.3 HDL-C Content in LiquidsDerived Formula:HDL-C (mmol/L) = (CStandard × V₂) × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ V₁ × DSimplified Formula:HDL-C (mmol/L) = CStandard × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) × D3.4 HDL-C Content in SerumDerived Formula:HDL-C (mmol/L) = (CStandard × V₂) × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ V₁ × 2 × DSimplified Formula:HDL-C (mmol/L) = 2 × CStandard × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) × D3.5 Based on Cell CountDerived Formula:HDL-C (nmol/10⁴ cells) = (CStandard × V₂) × 10³ × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) ÷ (500 × V₁ ÷ V) × DSimplified Formula:HDL-C (nmol/10⁴ cells) = 2 × CStandard × (ΔATest - ΔABlank) ÷ (ΔAStandard - ΔABlank) × DParameter Definitions:CStandard: Concentration as indicated on the label (mmol/L or µmol/mL)V₁: Volume of sample added (0.0025 mL)V: Volume of extraction buffer (ethanol) added (1 mL)V₂: Volume of standard added (0.0025 mL)D: Dilution factor (1 if not diluted)2: Dilution factor in serum pre-treatment500: Number of cells (in units of 10⁴)W: Sample weight (g)Cpr: Protein concentration of the supernatant (mg/mL); Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.Precautions1. It is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure. Based on the preliminary results, determine or adjust sample concentrations to prevent unnecessary waste of samples or reagents.2. This product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation... Read More | Products content Box 1: Circularization reagentC666001Component16 TStorageC666001ASplint Oligo20 µL-20℃.Avoid freeze/thaw cycle. C666001B5×Splint Buffer T4250 µL-20℃.Avoid freeze/thaw cycle. C666001CDNA Ligase50 µL-20℃.Avoid freeze/thaw cycle. C666001DDigestion Products content Box 1: Circularization reagentC666001Component16 TStorageC666001ASplint Oligo20 µL-20℃.Avoid freeze/thaw cycle. C666001B5×Splint Buffer T4250 µL-20℃.Avoid freeze/thaw cycle. C666001CDNA Ligase50 µL-20℃.Avoid freeze/thaw cycle. C666001DDigestion Buffer20 µL-20℃.Avoid freeze/thaw cycle. C666001EDigestion Enzyme I70 µL-20℃.Avoid freeze/thaw cycle. C666001FDigestion Enzyme III25 µL-20℃.Avoid freeze/thaw cycle. Box 2: Magnetic Beads for DNA Purification and RecoveryC666001Component16 TStorageC666001GCMPure4×1.5 mL2-8℃Products IntroductionThe Cyclization Kit is a modular kit tailored for the MGI high-throughput sequencing platform. With this kit, PCR products after junction ligation can be prepared into single-stranded circular DNA libraries suitable for MGI sequencers. All reagents provided in the kit have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction. Provide your own instruments, reagents and consumables1. Magnetic frame: DynaMagTM-2 (Cat. No. 12321D) is recommended.2. "Qubit" 3.0 Fluorescence Quantimeter (ThermoFisher, Cat. No. Q33216)3. Qubit" ssDNA Assay Kit (Invitrogen, Cat. No. Q10212)4. Anhydrous ethanol, EB (10 mM Tris-HCl, pH 8.0), NF Water (pH between 7.0 and 8.0).5. reaction tubes: low adsorption PCR tubes with 1.5 mIEP tubes are recommended: 5.Tip: It is recommended to use a high quality filter tip to prevent contamination of kits and libraries. Pre-experiment Preparation and Important Notes 1. Sample preparation.PCR product: 2330 ng total (total amount when multiple PCR products are mixed) in a volume of 49 pL (if the volume of PCR product is insufficient, add NF Water to bring the total volume to 49 pl). -PCR product: Fragment size: The fragment peak is between 200-500 bp. -PCR product fragment size: Fragment peaks between 200-500 bp. -PCR product modification: Fixed sequences (with Index) for MGISEQ-2000, MGISEQ-200 and BGISEQ-500 sequencing platforms were added.2. Reagent preparation-Remove the corresponding reagents from the kit, centrifuge briefly, and place the enzyme mixture on ice until ready to use: buffers need to be dissolved at room temperature before use, then centrifuged with shaking and placed on ice until ready to use, and NF Water and EB are placed at room temperature until ready to use: "Please make up the mixture on ice:Precipitation may appear after the buffer in the kit is dissolved, the precipitation does not affect the function of the reagent, please shake and mix well until the precipitation disappears and then use. Schematic diagram of the cyclization process procedurecyclize 1. 1 wl of Splint Oligo was added to the 49JI PCR product. The product was denatured and incubated on a PCR instrument at 95°C for 3 min, then immediately transferred to an ice bath and allowed to stand for 2 min. 2. The reaction mixture was prepared on ice according to the following system. 3. Add 15ul of the above reaction mixture to 50µl of denatured DNA.4. Place the above PCR tubes on the PCR instrument under the following conditions Reaction. digest 1. Prepare the digestion reaction solution on ice according to the following system. 2. After the cyclization reaction, add 8l of digestion reaction solution directly to the cyclization system, mix well, centrifuge briefly and then place the PCR tube on the PCR instrument and react under the following conditions. 3. Purification was carried out immediately after the reaction.Purification of digestive products1. Remove CMPure at room temperature 30 minutes prior to use and mix well with shaking.2. Transfer the digested product to a 1.5 mIEP tube, pipette 340 pICMPure into the digested product, mix well by gently blowing 10 times with a pipette and incubate for 10 minutes at room temperature.3. Instantaneous centrifugation, place the EP tube on a magnetic rack and let stand for 5 minutes until the liquid is clear, pipette and discard the supernatant.4. Keep the EP tube fixed on a magnetic rack, add 250ul of freshly prepared 80% ethanol, let it stand at room temperature for 1 minute, then carefully discard the supernatant.5. Repeat step 4 once, try to suck up the liquid at the bottom of the tube: Note: Do not suck up the magnetic beads, so as not to affect the yield.6. Keep the EP tube fixed on the magnetic rack, open the cap and dry it at room temperature for 5-10 minutes.7. Remove the EP tube from the magnetic rack, add 35ul of EB or NF Water for DNA elution, pipette blow to mix and dissolve at room temperature for 10 min.8. Centrifuge instantaneously, place the EP tube on a magnetic rack and let stand for 2 minutes until the liquid is clarified, transfer the supernatant to a new EP tube. -Store at 20C and leave to prepare DNB... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | DescriptionPhoto KitAlysis Starter Kit enables screening of 24 micro-scale simultaneous photocatalytic reactions with consistent and reproducible photon intensity. User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsComponents:Photo KitAlysis LED ControllerBlue LED DescriptionPhoto KitAlysis Starter Kit enables screening of 24 micro-scale simultaneous photocatalytic reactions with consistent and reproducible photon intensity. User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsComponents:Photo KitAlysis LED ControllerBlue LED Array (470 nm)Photo KitAlysis Reaction BlockTorque screwdriverSmall screwdriver to easily remove torqued screws after reaction is completeFeatures:Designed and tested by synthetic chemists.Controller provides repeatable milliamp selection for photon intensity0-30 mA variable LED output3 different LED options: blue (470 nm, included), green (527 nm, sold separately), and white (sold separately)Non-magnetic LED baseChemically resistant LED coverPTFE coated cablingDesigned to be used withPhoto KitAlysis High-Throughput Reaction Screening Kit(sold separately).Best when used withKitAlysis Benchtop Inertion Box(sold separately)... Read More | Product content: U665923Component50 T200 TStorageU665923ABuffer GTL15 mL60 mLRTU665923BBuffer GL15 mL50 mLRTU665923CBuffer GW1 (concentrate)13 mL52 mLRTU665923DBuffer GW2 (concentrate)15 mL70 mLRTU665923EBuffer GE15 mL60 mLRTU665923FProteinase K1.25 mL4×1.25 mLRTU665923GSpin Columns DM with Product content: U665923Component50 T200 TStorageU665923ABuffer GTL15 mL60 mLRTU665923BBuffer GL15 mL50 mLRTU665923CBuffer GW1 (concentrate)13 mL52 mLRTU665923DBuffer GW2 (concentrate)15 mL70 mLRTU665923EBuffer GE15 mL60 mLRTU665923FProteinase K1.25 mL4×1.25 mLRTU665923GSpin Columns DM with Collection Tubes50 EA200 EART Product Introduction:This reagent kit is suitable for extracting high-purity total DNA from various samples such as fresh or frozen animal tissues, cells, blood, bacteria, etc. This product can purify DNA fragments with a maximum molecular weight of 50 kb. The purification process does not require the use of toxic solvents such as phenol or chloroform, nor does it require ethanol precipitation. This reagent kit adopts an optimized buffer system to efficiently and specifically bind DNA from the lysis solution to the silica matrix centrifuge adsorption column. Inhibitors of PCR and other enzymatic reactions can be effectively removed through a two-step washing step. Finally, high-purity DNA can be obtained by washing with low salt buffer or water. The purified DNA can be directly used for downstream experiments such as enzyme digestion, PCR, Real Time PCR, library construction, Southern Blot, and molecular labeling.Self prepared reagent: anhydrous ethanolEnzymatic Lysis Buffer (preparation required for extracting genomic DNA from Gram positive bacteria).Self prepared reagent: Enzymatic Lysis Buffer Formula: 20 mM Tris, pH 8.0; 2 mM Na2 EDTA; 1.2% Triton self prepared reagent: X-100; Lysozyme with a final concentration of 20 mg/mL.Preparation and important precautions before the experiment:1. Samples should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume.2.If extracting the genome of bacterial cultures with a large accumulation of secondary metabolites or thick cell walls, it is recommended to collect samples early in the logarithmic growth phase.3.Before the first use, anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the reagent bottle label.4. Before use, please check if there is any crystallization or precipitation in Buffer GTL and Buffer GL. If there is any crystallization or precipitation, please dissolve Buffer GL and Buffer GTL again in a 56 ℃ water bath.5. If downstream experiments are sensitive to RNA contamination, 4 can be added before adding Buffer GL µ RNase A of L DNase Free (100 mg/mL) was not provided in this kit.Operation steps：Genome extraction from blood and cell samples1. Material processing1a If the extracted material is mammalian anticoagulant blood (non nucleated red blood cells), it can be directly directed to 50-200 µ Add Buffer GTL to fresh or frozen anticoagulant blood samples to supplement up to 200 µ L;1b If the extracted material is anticoagulant blood from poultry, birds, amphibians, or lower level organisms, and their red blood cells are nucleated cells, take 5-10 µ L fresh or frozen anticoagulant blood samples, add Buffer GTL to supplement up to 200 µ L;1c The cells cultured on the wall should be first processed into a cell suspension (with a maximum extraction amount of 5 × 10 cells), centrifuged at 2000 rpm (400 × g) for 5 minutes, discarded from the supernatant, and added with 200 µ L GTL, oscillate until the sample is completely suspended;Note: To remove RNA, add 4 after completing the above steps µ RNase A solution with a concentration of 100 mg/mL was vortexed for 15 seconds and left at room temperature for 2 minutes.2. Add 20 µ L Protein K.3. Add 200 µ L Buffer GL, vortex oscillation thoroughly mixed, 56 ℃ water bath for 10 minutes.4. Temporarily centrifuge to remove water droplets from the inner wall of the tube cover. Join 200 µ L anhydrous ethanol, vortex and shake thoroughly to mix well. Short centrifugation.Attention: 1) After adding Bu ff er GL and anhydrous ethanol, immediately vortex shake and mix well.2) The addition of Bu ff er GL and anhydrous ethanol may produce white precipitates, which will not affect subsequent experiments. Some organizations may form sol-gel products after adding Bu ff er GL and anhydrous ethanol, and it is recommended to perform severe shaking or vortex treatment at this time.5. Add all the solutions obtained in the previous step to the spin columns DM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm (~13400 × g) for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.6. Add 500 to the adsorption column µ L Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Add 500 to the adsorption column µ L Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.Note: To further improve DNA purity, repeat step 7.8.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).9. Place the adsorption column in a new centrifuge tube (provided by oneself) and add 50-200 to the middle of the adsorption column in the air µ L Buffer GE or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃.Attention:1) If downstream experiments are sensitive to pH or EDTA, they can be washed off with sterilized water. The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). When the pH value is below 7.0, the elution efficiency is not high.2) Preheating the GE in a water bath at 65-70 ℃ and incubating it at room temperature for 5 minutes before centrifugation can increase yield; Use an additional 50-200 µ Re washing with GE or sterilized water can increase yield.3) If the final concentration of DNA needs to be increased, the obtained solution can be re added to the adsorption column, left at room temperature for 2-5 minutes, and centrifuged at 12000 rpm for 1 minute; If the elution volume is less than 200 µ L. It is possible to increase the final concentration of DNA, but it may reduce the total yield. If the amount of DNA is less than 1 µ g. Recommended 50 µ Wash with GE or sterilized water.4) Because DNA stored in water is affected by acidic hydrolysis, if long-term preservation is required, it is recommended to elute with Bu ff er GE and store at -20 ℃.Genome extraction from animal tissues1. Material processingIf the extracted material is animal tissue, take 25 mg (the amount of spleen tissue should be less than 10 mg); If the material is mouse tail, take a section of rat tail with a length of 0.4-0.6 cm or two sections of mouse tail with a length of 0.4-0.6 cm.1a. After liquid nitrogen grinding or cutting the sample into small pieces, place it in a 1.5 mL centrifuge tube and add 180 mL µ Label different samples with L Buffer GTL.1b If using a homogenizer to process the sample, add no more than 80% of the homogenizer to the sample before homogenization µ L Buffer GTL, add 100 after homogenization µ L Buffer GTL.Attention:1) Ensure that the quantity of each organization does not exceed the recommended range.2) The tissue samples can be ground with liquid nitrogen or homogenized with a homogenizer before adding Bu ff er GTL, which can increase the cracking efficiency.2. Add 20 µ L Protein K, vortex oscillation thoroughly mixes the sample. Take a 56 ℃ water bath until the tissue is completely lysed. During the incubation process, the centrifuge tube can be inverted or shaken periodically to disperse the sample.Attention:1) The digestion time varies for different tissues, usually taking 1-3 hours to complete. The tail of the mouse needs to be digested for 6-8 hours, and if necessary, overnight digestion will not affect subsequent operations.2) If there is still gel like substance after incubation and vortex oscillation, extend the incubation time at 56 ℃ or add another 20 µ L Protein K digestion.3) To remove RNA, add 4 after completing the above steps µ RNase A solution with a concentration of 100 mg/mL, vortex for 15 seconds, and leave at room temperature for 5-10 minutes.3. Add 200 µ L Buffer GL, vortex shake thoroughly and mix well, take a water bath at 70 ℃ for 10 minutes. Add 200 after brief centrifugation µ L anhydrous ethanol, vortex and shake thoroughly to mix well.Attention:1) After adding Bu ff er GL and anhydrous ethanol, immediately vortex and shake to mix well.2) The addition of Bu ff er GL and anhydrous ethanol may produce white precipitates, which will not affect subsequent experiments. Some tissues (such as the spleen and lungs) may form sol-gel products after adding Bu ff er GL and anhydrous ethanol. In this case, it is recommended to perform vigorous shaking or vortex treatment.4. Centrifuge briefly and add all the solution obtained in step 3 to the spin columns DM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm (~13400 × g) for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.5. Add 500 to the adsorption column µ L Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.6. Add 500 to the adsorption column µ L Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.Note: To further improve DNA purity, repeat step 6.7.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).8. Place the adsorption column in a new centrifuge tube (provided by oneself) and add 50-200 to the middle of the adsorption column in the air µ L Buffer GE or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃.Attention:1) If downstream experiments are sensitive to pH or EDTA, they can be washed off with sterilized water. The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). When the pH value is below 7.0, the elution efficiency is not high.2) Preheating the GE in a water bath at 65-70 ℃ and incubating it at room temperature for 5 minutes before centrifugation can increase yield; Use an additional 50-200 µ Re washing with GE or sterilized water can increase yield.3) If the final concentration of DNA needs to be increased, the obtained solution can be re added to the adsorption column, left at room temperature for 2-5 minutes, and centrifuged at 12000 rpm for 1 minute; If the elution volume is less than 200 µ L. It is possible to increase the final concentration of DNA, but it may reduce the total yield. If the amount of DNA is less than 1 µ g. Recommended 50 µ Wash with GE or sterilized water.4) Because DNA stored in water is affected by acidic hydrolysis, if long-term preservation is required, it is recommended to elute with Bu ff er GE and store at -20 ℃. i ii Genomic extraction of blood and cell samples1. Bacterial sample pretreatment1a Gram negative bacteria(1) Take 1-5mL of bacterial culture (10 ^ -10 ^ cells, up to a maximum of 2 × 10 ^ cells) and place it in a centrifuge tube (self prepared). Centrifuge at 12000 rpm (~13400 × g) for 1 minute and try to aspirate the supernatant as much as possible.(2) Add 180 to the precipitate µ L Buffer GTL, shake to suspend bacterial weight.(3) Join 20 µ L Protein K, vortex mix well, incubate at 56 ° C until the bacterial cell is completely lysed, and during the incubation process, invert or shake the centrifuge tube periodically to disperse the sample.Note: To remove RNA, add 4 after completing the above steps µ L RNase A solution with a concentration of 100 mg/mL, shake well and let stand at room temperature for 5-10 minutes.(4) Join 200 µ L Buffer GL, vortex oscillation mixing.1b Gram positive bacteria(1) Take 1-5 mL of bacterial culture (10 ^ -10 ^ cells, maximum not exceeding 2 x 10 ^ cells) and place it in a centrifuge tube (self prepared). Centrifuge at 12000 rpm for 1 minute and try to aspirate the supernatant as much as possible.(2) Join 180 µ L Enzymatic Lysis Buffer (self provided) suspends the bacterial weight.(3) Incubate at 37 ℃ for 30 minutes.(4) Join 20 µ L Protein K vortex oscillation, thoroughly mixed. Join 200 µ L Buffer GL, vortex oscillation mixing. Incubate at 56 ℃ for 30 minutes.Attention:1) If necessary, incubation at 95 ° C for 15 minutes can inactivate the pathogen, but incubation at 95 ° C can cause some DNA degradation.2) To remove RNA, add 4 after completing the above steps µ L RNase A solution with a concentration of 100 mg/mL, shake well and let stand at room temperature for 5-10 minutes.2. Add 200 µ L anhydrous ethanol, vortex and shake thoroughly to mix well.Attention: Adding anhydrous ethanol may produce white precipitates, which will not affect subsequent experiments.3. Add all the solution obtained from step 2 (including the formed precipitate) to the adsorption column (Spin Columns DM) that has been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.4. Add 500 to the adsorption column µ L Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.5. Add 500 to the adsorption column µ L Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.Note: To further improve DNA purity, repeat step 5.6.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).7. Place the adsorption column in a new centrifuge tube (provided by oneself) and add 50-200 to the middle of the adsorption column in the air µ L Buffer GE or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃.Attention:1) If downstream experiments are sensitive to pH or EDTA, they can be washed off with sterilized water. The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). When the pH value is below 7.0, the elution efficiency is not high.2) Preheating the GE in a water bath at 65-70 ℃ and incubating it at room temperature for 5 minutes before centrifugation can increase yield; Use an additional 50-200 µ Re washing with GE or sterilized water can increase yield.3) If the final concentration of DNA needs to be increased, the obtained solution can be re added to the adsorption column, left at room temperature for 2-5 minutes, and centrifuged at 12000 rpm for 1 minute; If the elution volume is less than 200 µ L. It is possible to increase the final concentration of DNA, but it may reduce the total yield. If the amount of DNA is less than 1 µ g. Recommended 50 µ Wash with GE or sterilized water.4) Because DNA stored in water is affected by acidic hydrolysis, if long-term preservation is required, it is recommended to elute with Bu ff er GE and store at -20 ℃... Read More |