| Description | Inquire | Bacterial protein extraction reagents use mild non-ionic detergents and are suitable for extracting recombinant proteins expressed in Escherichia coli and insect cells. During the extraction process, there is no need for ultrasonic fragmentation, effectively avoiding contamination of exogenous Bacterial protein extraction reagents use mild non-ionic detergents and are suitable for extracting recombinant proteins expressed in Escherichia coli and insect cells. During the extraction process, there is no need for ultrasonic fragmentation, effectively avoiding contamination of exogenous proteins. This product can be applied to extract soluble proteins from bacterial lysates. The bacterial protein extraction kit adds a mixture of lysozyme, DNase I, and protease inhibitors to the extraction reagent, which can improve the efficiency of protein extraction and reduce the viscosity caused by DNA, effectively avoiding protein degradation. The extracted protein maintains biological activity and can be subjected to downstream operations such as IP, Western blot, and protein purification. Component B665764 100 preps Bacterial Protein Extraction Reagent 100 ml Protease Inhibitor Cocktail (100x) 1 ml Lysozyme （50 mg/ml） 200µl DNaseⅠ（1,000 U/ml） 100µl Notes:1. This product is suitable for extracting proteins from fresh or frozen bacterial and insect cells.2. This product uses Tris buffer system. Please use the same buffer system for protein purification after extraction.3. The protein lysis solution obtained from this product can be used for protein quantification using BCA or Bradford method.4. For special strains, if the extraction effect is not ideal, the sample can be frozen before protein extraction.5. Depending on the specific situation, protease inhibitors, salts, chelating agents, reducing agents, etc. can be added to this product.Operation steps: ● Insect cell protein extraction1. Collect cells by low-speed centrifugation. Add 10 to every 1 ml of Bacterial Protein Extraction Agent µ The Protein Inhibitor Cocktail is 1 x working fluid.2. Weigh the wet weight of the cells and add 1 x working solution at a rate of 10 ml/g.3. After resuspension, incubate on ice for 20 minutes (the ice storage time should be adjusted according to different cell types).Centrifuge at 4.15000 × g for 15 minutes to isolate soluble proteins. ● Extraction of soluble bacterial proteins 1. Centrifuge for 10 minutes at a rate of 5000 × g and collect the bacterial cells.2. Optional steps: Add 1 ml of Bacterial Protein Extraction Reagent every 1 ml µ DNase I (1000 U/ml), 2 µ Lysozyme (50 mg/ml) and 10 µ Protein Inhibitor Cocktail, vortex oscillation and mixing. 3. Add 20 ml of Bacterial Protein Extraction Reagent to each gram of bacterial precipitate, and add the extraction solution to the bacterial precipitate. Vortex thoroughly or use a pipette to blow up and down until the bacterial precipitate is completely resuspended.4. After resuspension, incubate at room temperature for 10-15 minutes (the storage time should be adjusted according to different cell types). 5. Centrifuge at 15000 × g for 5 minutes.6. Transfer the supernatant to a new centrifuge tube (the supernatant is soluble protein) for protein quantification and downstream experiments.Note: If the target protein exists in the form of inclusion bodies, inclusion body protein solution can be used for dissolution or expression conditions can be optimized to increase the expression of soluble proteins.Frequently asked questions: Problem Possible reasons Resolvent The target protein is insoluble The target protein is expressed as an inclusion body Optimize expression conditions or add Lysozyme and DNase I to protein extraction reagents using inclusion body protein solution After adding Lysozyme, the target protein has not been extracted yet Temperature too low Restore the reagent to room temperature After adding Lysozyme, the target protein has not been extracted yet Lysozyme Decreased or inactivated activity Add more Lysozymes or replace with new enzymes Extract has high viscosity DNase I Decreased or inactivated activity Add more DNase I or replace with a new DNase I to increase the final concentration of magnesium ions to 2 mM After protein extraction, most of the proteins still exist in the precipitate Excessive protein content Add Lysozyme and DNase I The protein extraction reagent has sediment precipitation Temperature too low Restore the protein extraction reagent to room temperature... Read More | Product Content D669986Component50 TStorageD669986ABuffer SA15 mLRTD669986B2×PCR MasterMix1 mL-20℃. Avoid freeze/thaw cycle.D669986CProteinase K12.5 mgRTD669986DProteinase K Storage Buffer1.25 mLRTProductsThis kit adopts a unique buffer system containing all the reagents for rapid Product Content D669986Component50 TStorageD669986ABuffer SA15 mLRTD669986B2×PCR MasterMix1 mL-20℃. Avoid freeze/thaw cycle.D669986CProteinase K12.5 mgRTD669986DProteinase K Storage Buffer1.25 mLRTProductsThis kit adopts a unique buffer system containing all the reagents for rapid preparation of genomic DNA and PCR amplification, and is suitable for one-step extraction of genomic DNA from various plant and animal tissues and bacteria and for PCR amplification. The whole extraction process does not require liquid nitrogen grinding, organic solvent extraction, anhydrous ethanol precipitation, and the quality of extracted DNA is stable. The 2×PCR MasterMix provided in this kit is a highly compatible PCR reagent that can amplify DNA samples efficiently and specifically, which includes DNA polymerase, dNTPs, MgCl2, reaction buffer, PCR reaction enhancer and so on. It is characterized by fast and easy, high sensitivity, high specificity, good stability, etc. It is especially suitable for high throughput screening.Pre-experiment Preparation and Important Notes1. Add the specified amount of Proteinase K Storage Buffer to Proteinase K to dissolve it and store it at -20℃. Do not leave the prepared Proteinase K at room temperature for a long time, and avoid repeated freezing and thawing to avoid affecting its activity.2. Repeated freezing and thawing of the samples should be avoided, as this will result in smaller DNA fragments and a decrease in the amount of extracted DNA.3. Before use, please check Buffer SA for crystallization or precipitation. If crystallization or precipitation occurs, please re-dissolve Buffer SA in a 56℃ water bath.4. The PCR MasterMix provided with this product is 2×, when using it, you need to add template and primer, and add RNase-Free Water to make up the volume, so that its concentration is 1× to carry out the reaction.Procedure1. Fetch:Plant material: take about 10 mg of sample in a centrifuge tube (provided); Animal material: take about 10 mg of sample in a centrifuge tube (provided);Bacteria: Take 200-800 µL of bacteria in good growth condition in a centrifuge tube (self-provided) and collect the bacteria.2. Add 200 µL of Buffer SA and vortex to mix.Note: In the case of plant leaves and animal tissues, they should be ground with a pestle and mortar as much as possible: in the case of plant seeds, they should be crushed and finely ground beforehand; bacterial and 1-3 mm rat-tail samples can be directly vortex lysed.3. Add 10µL of Proteinase K, mix well, incubate at 56℃ for 10 minutes, and treat at 95℃ for 5 minutes.Note: 1) In the case of animal tissue samples, the incubation time at 56°C may be extended to 30 minutes as appropriate; if there is any incompletely digested tissue, it should be removed as thoroughly as possible after centrifugation in the next step.2) Be careful not to exceed 5 minutes when treating at 95°C.4. 13,000 rpm (~17,900 x g), centrifugation for 5 minutes.5. Transfer the supernatant to a new centrifuge tube (self-prepared) and use it directly for PCR amplification, or store the solution at 4℃ or -20℃.6. PCR amplification:1) PCR reaction system:The following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.reagents20 µL systemfinal concentration2×PCR MasterMix10 µL1×Forward Primer, 10 µM1 µL0.4 µMReverse Primer, 10 µM1 µL0.4 µMTemplate DNA1-2 µL RNase-free Waterup to 20 µLNote: Please use the final concentration of 0.2-0.6µM as a reference for setting the range of primer concentration. If the amplification efficiency is not high, the concentration of primer can be increased; if a non-specific reaction occurs, the concentration of primer can be decreased, thus optimizing the reaction system.2）PCR reaction conditions:movetemptimingpremutability94°C2mindenaturation94°C30sannealing (metallurgy)55-65°C30s30-40 cyclesreach72°C60sultimate extension72°C5minNote: 1) In general, the annealing temperature is 5℃ lower than the melting temperature of the amplification primer Tm, and the annealing time is generally 30-60 seconds. When the desired amplification efficiency cannot be obtained, the annealing temperature should be lowered appropriately; when a non-specific reaction occurs, the annealing temperature should be raised, thus optimizing the reaction conditions.(2) The extension time is set according to the size of the fragment to be amplified, and the amplification efficiency of Taq DNA Polymerase included in this product is 1kb/30s. 3) The number of cycles can be set according to the downstream application of the amplification product. If the number of cycles is too low, the amplification is insufficient; if the number of cycles is high, the chance of mismatch will increase and the non-specific background will be serious. Therefore, the number of cycles should be minimized under the premise of ensuring the product yield.(3) Result detection: 5 µL of reaction product was taken at the end of the reaction and directly detected by agarose gel electrophoresis... 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 | Products contentProducts IntroductionThe Single Cell Whole Genome Amplification Kit can be used as a template for whole genome amplification of single cells or micro samples. The total time for single-cell amplification is about 3 hours, and 2-5 µg of genomic DNA, with a size of 200-1500 bp, Products contentProducts IntroductionThe Single Cell Whole Genome Amplification Kit can be used as a template for whole genome amplification of single cells or micro samples. The total time for single-cell amplification is about 3 hours, and 2-5 µg of genomic DNA, with a size of 200-1500 bp, can be obtained after lysis, pre-amplification and amplification. The amplified product can be widely used in second-generation sequencing, large fragment copy number variation analysis, SNP typing, qPCR analysis and gene chip analysis.Bring your own instruments and reagentsPCR instrument Reaction tubes: low adsorption tubes recommended Gun Heads: High quality filtered gun heads are recommended Microcentrifuge, vortex mixercaveat The sensitivity of this product is very high, the experimental operation should be completed in a positive pressure ultra-clean bench or clean environment, the concentration of the amplification reaction products is high, should be well isolated to avoid aerosol contamination caused by amplification products.Operation flow diagramprocedurePre-experiment preparationSingle cells were obtained by flow cytometry sorting, buffer dilution, micromanipulation and laser microdissection. It is recommended that the cells be washed prior to the experiments with a 1× PBS solution free of Mg2+ and Ca2+, taking care to ensure that the volume of PBS solution in subsequent experiments does not exceed 2 µl. take note of Since the whole experiment is carried out in the same PCR tube and the reaction volume is small, the pipette tip should not touch the liquid in the tube when adding liquid, so as to avoid taking single cells or DNA out of the reaction system; when pipetting, please add the liquid along the wall of the tube carefully and do not blow the liquid in the PCR tube; before the reaction, please centrifuge briefly to make sure that the liquid in the reaction system is mixed evenly. Thaw the cell lysate, pre-amplifier and amplifier on ice before use.cell lysis 1）Mix Cell Lysis Buffer and Cell Lysis Enzyme according to the number of reactions N, shake to mix, centrifuge briefly and set aside.2）Mix single cells with the cell lysis mix in a PCR tube and run the following program.2. Pre-amplification reaction1）Mix Cell Lysis Buffer and Cell Lysis Enzyme according to the number of reactions N, shake to mix, centrifuge briefly and set aside.2）Add 5 µl of pre-amplification mix to 10 µl of lysis reaction product from the previous step and run the following program. 3. Amplification reaction1）Mix Amplification Buffer and Amp Enzyme Mix according to the number of reactions N, mix with shaking, centrifuge briefly and set aside.2）Add 60 µl of amplification mix to 15 µl of pre-amplification reaction product from the previous step and run the following program.Note: The number of cycles can be adjusted as needed, 14 cycles are recommended for single cells obtained by flow sorting, etc.Amplification product detection 1. Agarose gel electrophoresis 5 µl of the amplified product was subjected to agarose gel electrophoresis (1% agarose gel, 110 V, 25-35 min), and the amplified product was 200-1500 bp in size. 2. Quantitative Amplification products were subjected to magnetic bead or column purification, and purified products were quantified using Qubit with a final yield of 2-5 µg... Read More |