| Description | Product Introduction:Due to the low stoichiometry of phosphorylation, the concentration of phosphopeptides is a key step for the successful implementation of phosphoproteomics experiments. PolyMAC provides an effective and significantly improved method to achieve more complete enrichment of Product Introduction:Due to the low stoichiometry of phosphorylation, the concentration of phosphopeptides is a key step for the successful implementation of phosphoproteomics experiments. PolyMAC provides an effective and significantly improved method to achieve more complete enrichment of phosphopeptides. This highly selective enrichment method can be used for most complex samples because it offers optimal specificity and recovery rate. PolyMAC is a polymer-based immobilized metal ion affinity capture method with excellent phosphoproteome coverage and recovery rate. It is a soluble nanopolymer designed to interact with phosphopeptides in solution, and then be captured on a solid-phase carrier for washing and phosphopeptide elution. Compared with the commonly used titanium dioxide and immobilized metal ion affinity capture methods, PolyMAC has better reproducibility and enrichment of phosphorylated peptides, with the selectivity of phosphorylated peptides close to 95% and the recovery rate > 90%. Importantly, PolyMAC has very good recovery rate and selectivity for samples with low total protein phosphorylation levels.Experimental Flowchart of Phosphopeptide Protein (Peptide Fragment) Extraction Kit (P1456460)Product Components and Storage Conditions:P1456460Component24T48T96TStorageP1456460APolyMAC Beads0.75 mL1.5 mL3 mL4℃P1456460BPolyMAC Loading Buffer25 mL50 mL100 mLRTP1456460CPolyMAC Washing Buffer I25 mL50 mL100 mLRTP1456460DPolyMAC Washing Buffer II25 mL50 mL100 mLRTP1456460EPolyMAC Elution Buffer3.75 mL7.5 mL15 mLRTP1456460FPolyMAC Tips24T48T96TRTProduct Features:Easy to operate: Phosphopeptides can be prepared quickly, requiring only a metal bath and a conventional centrifuge.High stability: Strict quality inspection is conducted for each batch, ensuring high reproducibility of experimental results.Operating Procedure:1.Add 200 µL of Loading Buffer to the dried sample (50 µg - 100 µg) after enzymatic hydrolysis and desalting, and fully resuspend the sample.2.Vortex the PolyMAC Beads thoroughly (for 10 - 20 seconds), then take 25 µL into a 1.5 mL centrifuge tube, centrifuge instantaneously for 2 - 3 seconds, and then remove the upper storage solution. (The Beads settle quickly, so the operation should be fast when taking them.)3.Add the resuspended sample to the centrifuge tube containing PolyMAC Beads, then place it on a mixer, and shake vigorously at 26°C, >1200 rpm for 25 minutes.4.Add the sample to PolyMAC - Tips, first centrifuge at 20 g for 2 minutes, then centrifuge at 100 g for 1 minute to ensure that the sample solution flows out from the tip of the Tips into the centrifuge tube. (The rotation speed should not be too high, and the operation should be fast.)5.Wash once with 200 µL of Loading Buffer: first centrifuge at 20 g for 2 minutes, then centrifuge at 100 g for 1 minute, and discard the filtrate. Repeat this step 3 times. (The rotation speed should not be too high, and the operation should be fast.)6.Wash once with 200 µL of Washing Buffer I: first centrifuge at 20 g for 2 minutes, then centrifuge at 100 g for 1 minute, and discard the filtrate. Repeat this step 3 times. (The rotation speed should not be too high, and the operation should be fast.)7.Wash once with 200 µL of Washing Buffer II: first centrifuge at 20 g for 2 minutes, then centrifuge at 100 g for 1 minute, and discard the filtrate. Repeat this step 3 times. (The rotation speed should not be too high, and the operation should be fast.)8.Put the Tips into a new centrifuge tube to collect the eluted phosphopeptides. Add 50 µL of Elution Buffer to the Tips, centrifuge at 20 g for 2 minutes, then add another 50 µL of Elution Buffer, first centrifuge at 20 g for 2 minutes, then centrifuge at 100 g for 1 minute, and collect the filtrate. (The rotation speed should not be too high, and the operation should be fast.)Note: In steps 4 - 8, if the liquid flows down slowly, the rotation speed can be increased appropriately, gradually increasing to 50 g, and do not increase too much at one time!9.If there is any solution remaining in the pipette tip, push it out into the collection tube with a pipette, lyophilize it, and store it at -80°C for mass spectrometry detection.Precautions:This product is only for scientific research use by professionals, and shall not be used for clinical diagnosis or treatment, nor for food or drugs... Read More | Description：Acetylcholinesterases (AChEs) are enzymes that hydrolyze the neurotransmitter acetylcholine (ACh) to acetate and choline. AChE is located at the synaptic cleft and functions to terminate synaptic transmission by catalyzing the breakdown of ACh allowing cholinergic neurons to Description：Acetylcholinesterases (AChEs) are enzymes that hydrolyze the neurotransmitter acetylcholine (ACh) to acetate and choline. AChE is located at the synaptic cleft and functions to terminate synaptic transmission by catalyzing the breakdown of ACh allowing cholinergic neurons to return to a resting state after activation. It is also found in membranes of red blood cells, motor and sensory fibers, muscles, nerves and central and peripheral tissues. Changes in AChE activity may result from exposure to certain insecticides, which act as cholinesterase inhibitors. Inhibitors of AChE are also used to treat certain conditions such as dementia.Acetylcholinesterase activity assay kit has been used to determine the activity of acetylcholinesterase in a rat organophosphate model and in brain tissue homogenates.Principle：Acetylcholinesterase can catalyze the hydrolysis of acetylcholine to choline, and the reaction of choline with disulfide p-nitrobenzoic acid to produce 5-merhydryl-nitrobenzoic acid (TNB). The product has a characteristic absorption peak at 412 nm, and the activity of acetylcholinesterase can be characterized by the change of light absorption valueThe Dilution Calculator EquationConcentration (start)xVolume (start)= Concentration (final)× Volume (final)This equation is commonly abbreviated as: C1V1 = C2V2... Read More | Product introduction：Dualucif The firefly & Renilla assay kit (dual luciferase reporter assay kit) provides an effective means to detect the expression of genes. In DLR detection, the activities of firefly luciferase and Renilla luciferase can be detected in a single sample in turn. FirstProduct introduction：Dualucif The firefly & Renilla assay kit (dual luciferase reporter assay kit) provides an effective means to detect the expression of genes. In DLR detection, the activities of firefly luciferase and Renilla luciferase can be detected in a single sample in turn. First, luciferin was used as substrate to detect the activity of firefly luciferase, then substances inhibiting the catalysis of firefly luciferase were added, and coelenterazine was added to detect the activity of Renilla luciferase to achieve dual luciferase reporter gene detection. The bioluminescence system of luciferase and its substrate can detect gene expression very sensitively and efficiently. Usually, the transcriptional regulatory element or 5'promoter region of the gene of interest is cloned upstream of luciferase, or the 3'-utr region is cloned downstream of luciferase to construct a reporter gene plasmid, and then transfect the cells. After the cells are treated with appropriate drugs, the cells are lysed, and the transcriptional regulation effect of drug treatment on the target gene is judged by detecting the luciferase activity. Renilla luciferase is more often used as an internal reference for detecting transfection efficiency to eliminate the difference in cell number and transfection efficiency. Firefly luciferase is a protein with a molecular weight of about 61 kDa. In the presence of ATP, magnesium ions and oxygen, it can catalyze the production of oxyluciferin from luciferin. In the process of luciferin oxidation, it will produce a light signal. Renilla luciferase is a protein with a molecular weight of about 36 kDa. In the presence of oxygen, it can catalyze the oxidation of coelenteramide to coelenteramide, and also produce light signals in the process of coelenteramide oxidation. The optical signal of this kit can be measured by chemiluminescence instrument, microplate reader or liquid scintillation tester. The kit has the characteristics of rapid detection, high sensitivity, wide detection range and no interference of cell endogenous activity.Instruction：1.Cell lysis ( 1 ) Remove the medium and gently wash twice with PBS ( adherent cells can be operated directly, suspension cells need to be centrifuged to collect cells ). Add 1 × Lysis Buffer ( diluted component A with sterile water at 4 : 1 ) according to the following scheme, and then place the culture plate on a micro-oscillator at room temperature for 15 min to fully lyse the cells. Note : The pyrolysis products can be stored at room temperature for 6 h, and can be stored at − 70 °C for a long time ( the pyrolysis products cannot be repeatedly frozen and thawed ). ( 2 ) The pyrolysis products were centrifuged at 10000-15000 rpm for 3-5 min. After centrifugation, the supernatant was transferred into a new EP tube for subsequent detection. Note : Cells can be detected immediately after lysis, or frozen, and re-detected when needed. The frozen samples need to be thawed to room temperature for detection. 2. Preparation of working fluid ( 1 ) Restore all components to room temperature. ( 2 ) Dilute component C with component B to 0.2 mg / mL firefly luciferase working solution. Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the amount of a single experiment is small, it is recommended to be subpackaged into small specifications according to a single amount of use. ( 3 ) The E component was diluted into the renilla luciferase working solution with the D component, and the dilution method was 1 µL E component was added to the 49 µL D component. Note : Renilla luciferase working solution needs to be prepared now. 3.chemiluminescence value detection ( 1 ) According to the operation instructions of the instrument, the instrument with chemiluminescence detection function was opened, such as multifunctional microplate reader. The parameters were set, the determination time was 10 s, and the determination interval was 2 s. ( 2 ) each sample determination, take the sample 20-100 µL ( if the sample volume is enough, please add 100 µL ; if the sample amount is insufficient, the amount can be appropriately reduced, but the amount of detection holes needs to be consistent ). 1 × Lysis Buffer was blank control. ( 3 ) 100 µL firefly luciferase working solution was added to determine the RLU ( relative light unit ) value ( it is recommended that the microplate reader set up the Shaking mixing function ). Note : Since the luminescence is instantaneous, it is recommended to detect immediately after adding the firefly luciferase working solution. ( 4 ) 100 µL renilla luciferase working solution was added to determine the RLU ( relative light unit ) value ( Shaking mixing function is recommended for microplate reader ). ( 5 ) In the case of renilla luciferase as an internal reference, the RLU value measured by firefly luciferase was divided by the RLU value measured by renilla luciferase. According to the obtained ratio, the activation degree of the target reporter gene between different samples was compared. If firefly luciferase is used as an internal reference, similar calculations can also be performed.Component：Recommendation：It is recommended to use component B in advance to prepare 2 mg / mL storage solution, component B, component D and component C prepared as storage solution, and to carry out small batch packing according to the experimental requirements. All test working fluids are recommended to be used now to avoid repeated freezing and thawing.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. in order to obtain the best determination effect, when using a single tube chemiluminescence instrument for determination, the time from the mixing of sample and determination reagent to the pre determination should be controlled as much as possible; When using a multi-functional fluorescent microplate reader with chemiluminescence detection function, it is advisable to add all samples first, and then uniformly add firefly luciferase detection reagent. 3. the strongest wavelength of firefly luciferase catalyzed bioluminescence is 560 nm, and the strongest wavelength of Renilla luciferase catalyzed bioluminescence is 480 nm. 4. to prevent interference between holes, it is recommended to use white opaque orifice plate. 5. due to the influence of temperature on enzyme reaction, the sample and reagent should be measured after reaching room temperature. 6. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Study on gene expression regulation and promoter... Read More | Products contentNote: The amount of individual primers used is 1 µl, each N7-end primer can perform 10 DNA library constructs, and each kit can perform 240 DNA library constructs.Products IntroductionThis kit is a companion kit to the transposase-based Rapid DNA Library Construction Kit for Products contentNote: The amount of individual primers used is 1 µl, each N7-end primer can perform 10 DNA library constructs, and each kit can perform 240 DNA library constructs.Products IntroductionThis kit is a companion kit to the transposase-based Rapid DNA Library Construction Kit for Illumina platform library construction. Each kit contains one N5 primer and 24 N7 primers, which can be used to prepare 24 different single-ended Index libraries. All reagents provided in the kits have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction. The libraries can be used for sequencing on Illumina platforms such as HiSeq X-10/4000/2500/2000 and MiSeq. Provide your own instruments, reagents and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use Kangwei DNA purification and recovery kit by magnetic bead method.3. DNA building kit: It is recommended to use the Kangwei Century transposase method second-generation sequencing rapid DNA building kit.4. Anhydrous ethanol.5. Reaction tubes: It is recommended to use low adsorption PCR tubes with 1.5 ml centrifuge tubes; Tip: It is recommended to use a high quality filter tip to prevent contamination of kits and library samples. Pre-experiment Preparation and Important NotesPlease centrifuge briefly before opening the cap so that the liquid collects at the bottom of the tube to avoid cross-contamination between different primers. procedureFor the use of the CombiVision Second Generation Sequencing Multisample Primer Kit, please follow the CombiVision Second Generation Sequencing Rapid DNA Library Kit protocol. Index N501 Primer for Illumina Index N901-N996 Primer for Illumina... 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 |