| Description | DescriptionProvides an inert environment to run oxygen sensitive cross-coupling reactions in a laboratory fume hood.Designed to be used with KitAlysis High-Throughput Screening Kits and KitAlysis 24-Well Reaction Block | 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 | Inquire | 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 | Products contentProducts IntroductionThis kit is suitable for simple, rapid and efficient isolation and purification of DNA/RNA from whole blood, tissue homogenates, swabs, serum, plasma and other cell-free body fluids, etc. The unique buffer system enables the viral nucleic acids in the lysate to Products contentProducts IntroductionThis kit is suitable for simple, rapid and efficient isolation and purification of DNA/RNA from whole blood, tissue homogenates, swabs, serum, plasma and other cell-free body fluids, etc. The unique buffer system enables the viral nucleic acids in the lysate to bind to the silica gel centrifugal adsorbent columns in a highly efficient manner, and the viral nucleic acids obtained are of high purity and stable quality, free of protein, nuclease and other impurities, and can be used in a variety of routine operations, including PCR, fluorescence quantitative PCR and other experiments. It can be used for a variety of routine operations, including PCR, fluorescence quantitative PCR and other experiments.Bring your own instrumentsThermostatic mixer.Pre-experiment Preparation and Important Notes1. Read these instructions carefully before experimenting.2. If Proteinase K is to be stored for a long period of time, please keep it at -20℃.3. Check Buffer RLC for crystallization or precipitation prior to use, and if crystallization or precipitation occurs, redissolve Buffer RLC in a 56°C water bath.4. Pre-treatment of tissue samples: Take 20 mg of tissue samples into 1.5 mL centrifuge tubes (self-provided), add 500 µL of Buffer RLC, and after the tissue homogenizer breaks up, centrifuge the samples for 1 minute at 12,000 rpm (~13,400×g), and take 200 µL of supernatant as samples. procedure1. Take a 1.5 mL centrifuge tube (provided), add 500 µL of Buffer RLC, 200 µL of sample, 20 µL of Proteinase K, vortex for 5 s, and then place it in a thermostatic mixer at 1200 rpm for 10 min at room temperature. Note: For wet swab samples, 200 µL of sample was taken after sufficiently shaking and mixing. Note: For wet swabs, 200 µL was taken from the sample after it was soaked in 400 µL of saline, shaken and mixed thoroughly for 5 minutes, and then centrifuged at 12,000 rpm for 1 minute, and 200 µL was taken for extraction.2. Instantly remove the centrifuge tube and add the solution from step 1 to the Spin Columns DM in the collection tube. centrifuge at 12,000 rpm (~13,400 x g) for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.3. Add 500 µL of Buffer PGWT to the adsorbent column, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.4. Add 500 µL of Buffer GWT2 to the adsorbent column, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.5. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Place the adsorption column at room temperature for 2 minutes and allow to dry.6. Place the column in a new collection tube (RNase-Free Centrifuge Tube), add 40-100 µL of RNase-Free Water to the center of the column membrane, let it stand at room temperature for 2 minutes, and then centrifuge at 12,000 rpm for 1 minute to collect the nucleic acid solution. Store at -80℃ for a long time... Read More |