| Description | Inquire | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Product contentN666081Component50 TStorageN666081ANc-Buffer A50 mL2-8℃N666081BNc-Buffer B3 mL2-8℃N666081CNc-Buffer C25 mL2-8℃N666081DProtease Inhibitor Cocktail750 µL-20℃. Avoid freeze/thaw cycle.ProductsThe Nc-Nucleus/Plasma Protein Extraction Kit is a simple and rapid Product contentN666081Component50 TStorageN666081ANc-Buffer A50 mL2-8℃N666081BNc-Buffer B3 mL2-8℃N666081CNc-Buffer C25 mL2-8℃N666081DProtease Inhibitor Cocktail750 µL-20℃. Avoid freeze/thaw cycle.ProductsThe Nc-Nucleus/Plasma Protein Extraction Kit is a simple and rapid method for extracting nucleus and plasma proteins from mammalian cells and tissues, and the extracted proteins remain biologically active. The kit first cleaves the cell membrane and releases plasma proteins using the plasma protein extraction reagent, and then centrifuges the nucleus to obtain a nucleus precipitate. Finally, the nuclear proteins are extracted by the nuclear protein extraction reagent. The extracted nuclear and plasma proteins are of high purity, effectively avoiding cross-contamination of nuclear and plasma proteins, and can be used for subsequent operations such as Western, Gel Shift, reporter gene detection and enzyme activity determination.Caveat1. If phosphorylated proteins are to be extracted, add a phosphatase inhibitor to the extraction reagent.2. All sample handling should be done on ice.3. The amount of reagents can be adjusted according to the specific experimental situation to ensure that the ratio of each reagent used is Nc-Buffer A:Nc-Buffer B:Nc-Buffer C = 100:5.5:50.4. Higher speeds can be used for centrifugation.ProcedureI Extraction of cytoplasmic and cytosolic proteins from cells1. Please remove the extraction reagents Nc-Buffer A and Nc-Buffer C for pre-cooling before protein extraction.2. Collect the cells and count them. Centrifuge to remove supernatant.3. 1×107 cells were added with 1 ml of Nc-Buffer A (added to Protease Inhibitor Cocktail at a ratio of 1:99 within 2-3 minutes prior to protein pumping), vortexed for 5 seconds to mix well, and incubated on ice for 20 minutes.Note: The characteristics of various cells are different, and the amount of Nc-Buffer A needs to be adjusted according to the characteristics of different cells. If the protein concentration is small, reduce the amount of Nc-Buffer A and subsequent Nc-Buffer B and Nc-Buffer C proportionally.4. Add 55 µl of Nc-Buffer B, vortex for 5 seconds to mix thoroughly, and incubate on ice for 1 minute.5. Centrifuge at 12,000 rpm (~13,400 x g) for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytoplasmic protein).6. Add 500 µl of Nc-Buffer C (add Protease Inhibitor Cocktail at a ratio of 1:99 before use) to the precipitate obtained in the previous step, vortex for 5 seconds to mix thoroughly, resuspend the precipitate and incubate on ice for 40 minutes, vortexing and mixing at 10-minute intervals for about 15-30 seconds each time.7. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is for cytosolic proteins).II Extraction of cytoplasmic and cytosolic proteins from tissues1. Sampling and preservation of tissues.2. Remove the extraction reagents Nc-Buffer A and Nc-Buffer C for pre-cooling before protein extraction.3. Weigh the tissue and add 1 ml of Nc-Buffer A per 100 mg of tissue (add Protease Inhibitor Cocktail 2-3 minutes before protein extraction at a ratio of 1:99), homogenize well on ice with a homogenizer, and incubate on ice for 20 minutes.Note: The characteristics of various tissues are different, and the amount of Nc-Buffer A needs to be adjusted according to different tissues. If the protein concentration is small, reduce the amount of Nc-Buffer A and subsequent Nc-Buffer B and Nc-Buffer C proportionally.4. Add 55 µl of Nc-Buffer B, vortex for 5 seconds to mix thoroughly, and place on ice for 1 minute of incubation.5. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytoplasmic protein).6. Add 500 µl of Nc-Buffer C (add Protease Inhibitor Cocktail at a ratio of 1:99 before use) to the precipitate obtained in the previous step, vortex for 5 seconds to mix thoroughly, resuspend the precipitate and incubate on ice for 40 minutes, vortexing and mixing at 10-minute intervals at, each time for about 15-30 seconds.7. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytosolic protein)... Read More | Inquire | Product content: S665546Component50 TStorageS665546ABuffer QSL45 mLRTS665546BBuffer RIL11 mL2-8℃S665546CBuffer ML10 mLRTS665546DBuffer GW1 (concentrate)13 mLRTS665546EBuffer GW2 (concentrate)26 mLRTS665546FBuffer EBL13 mLRTS665546GRNase A240 µLRTS665546HLysis Tubes Ⅱ50 Product content: S665546Component50 TStorageS665546ABuffer QSL45 mLRTS665546BBuffer RIL11 mL2-8℃S665546CBuffer ML10 mLRTS665546DBuffer GW1 (concentrate)13 mLRTS665546EBuffer GW2 (concentrate)26 mLRTS665546FBuffer EBL13 mLRTS665546GRNase A240 µLRTS665546HLysis Tubes Ⅱ50 EARTS665546ISpin Columns DM With Collection Tubes50 EARTProduct IntroductionThis kit provides a method for extracting total DNA from soil or fecal samples, including the total DNA of cells, bacteria, parasites, and viruses in the samples. It is also suitable for extracting DNA from samples containing high concentrations of PCR reaction inhibitors. This reagent kit adopts a unique buffering system to efficiently bind DNA from the lysis solution to the adsorption column. Inhibitors of PCR and enzyme reactions, as well as residual impurities, can be effectively removed through washing steps. 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 second-generation sequencing (16S amplicons and metagenomes), library construction, PCR, qPCR, Southern Blot, enzyme digestion molecular markers, etc.Self prepared reagents1. Constant temperature mixer - Product number: CW25932. Anhydrous ethanol, isopropanol3. Vortex oscillator or tissue grinderPreparation and important precautions before the experiment1. Samples should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume.2.Before the first use, anhydrous ethanol should be added to Buffer GW1 (concentrate) and Buffer GW2 (concentrate) according to the instructions on the reagent bottle label.3. Take out the buffer RIL before use and store it at 2-8 ℃ immediately after use.Operation steps1. Centrifuge the Lysis Tube briefly to allow the beads to settle at the bottom.2. a. Add 0.1-0.3 g of soil or fecal sample to Lysis Tube, and add 740-820 µ L Buffer QSL and 4 µ L RNase A, tighten the tube cover and briefly vortex to mix.b. If fecal samples are stored in non lytic fecal preservation solutions (such as CWY041S and CWY041M), add 200 to Lysis Tube µ L-600 µ L solid-liquid mixture, centrifuge at 13000 rpm for 1 minute, discard the storage solution (if the amount of solid after centrifugation is too small, it can be enriched again, but should not exceed 0.3g). Join 620 µ LBuffer QSL and 4 µ L RNase A, tighten the tube cover and briefly vortex to mix.3. Fix the Lysis Tube in an oscillating grinding device equipped with a 2 mL adapter and process it according to the optimized grinding conditions of your equipment (see appendix).4. Shake the Lysis Tube on a constant temperature mixer at 70 ℃ and 1200 rpm for 10 minutes. Subsequently, centrifuge at 13000 rpm for 2 minutes to precipitate solid particles. Transfer 540 µ Transfer the supernatant to a new 2 mL centrifuge tube.5. Add 180 µ L Buffer RIL, vortex for 5 seconds, centrifuge at 13000 rpm for 2 minutes.Attention: Remove the buffer RIL before use and store it at 2-8 ℃ immediately after use.6. Add 160 to the new centrifuge tube in sequence µ L Buffer ML, 480 µ Supernatant from step 5, 320 µ L isopropanol, vortex for 5 seconds.7. Transfer the solution from the previous step to 650 µ Centrifuge at 12000 rpm (~13400 × g) for 1 minute into the spin columns DM that have been loaded into the collection tube.8. Discard the waste liquid in the collection pipe and place the adsorption column back into the collection pipe. Repeat step 7 until all the solution has been transferred.9. 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.10. 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. 11. Repeat step 10.12.12000 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.).13. Place the adsorption column in a new centrifuge tube (self provided) and add 50-200 drops of suspended droplets to the middle of the adsorption column µ L Buffer EBL 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 ℃.Note: 1) Incubating at room temperature for 5 minutes before centrifugation can increase yield.2) Use an additional 50-100 µ Further elution with L buffer or sterilized water can increase yield.3) If you want to increase the final concentration of DNA, you can add the DNA eluent obtained in step 13 back onto the adsorption membrane and repeat step 13, but it may reduce the total yield.4) The elution buffer does not contain chelating agents, please store DNA at -20 ℃.5) The residual trace PCR inhibitors in the genomic DNA template may have adverse effects on the PCR reaction, which can usually be resolved by diluting the DNA by 2-10 times.Appendix: Grind the sample using one of the following methods1. Manually vortex oscillate at maximum speed on the vortex oscillator for 10 minutes.2. On a vortex oscillator equipped with a 1.5-2 mL horizontal centrifuge tube holder, oscillate at maximum speed for 10 minutes (keeping the Lysis Tube horizontal). If the sample size exceeds 12, extend by 5-10 minutes. For example, using Scientific Industries or Mobile's Vortex Genie2 vortex oscillator.3.When using Qiagen's TissueLyser II, grind at 25Hz for 10 minutes.4.When using Qiagen's PowerLyzer 24 Homogenizer, homogenize at 2000 rpm for 30 seconds, pause for 30 seconds, and then homogenize again at 2000 rpm for 30 seconds.5.When using FastPrep-24 from MP Biomedicals, the recommended speed is 6.0 and the time is 40 seconds... Read More |