| Description | Inquire | G665573 Component 10 T Storage G665573A Buffer P1 30 mL RT G665573B Buffer P2 30 mL RT G665573C Buffer E3 30 mL RT G665573D Buffer PS 15 mL RT G665573E Buffer PW (concentrate) 10 mL RT G665573F Endo-Free Buffer EB 30 mL RT G665573G RNase A (10 mg/mL) 600 碌L RT G665573H Endo-Remover FX 10 EA G665573 Component 10 T Storage G665573A Buffer P1 30 mL RT G665573B Buffer P2 30 mL RT G665573C Buffer E3 30 mL RT G665573D Buffer PS 15 mL RT G665573E Buffer PW (concentrate) 10 mL RT G665573F Endo-Free Buffer EB 30 mL RT G665573G RNase A (10 mg/mL) 600 碌L RT G665573H Endo-Remover FX 10 EA RT G665573I Plungers 10 EA RT G665573J Spin Columns DX with Collection Tubes 10 EA RT G665573K Centrifuge Tubes (15 mL) 10 EA RTProduct IntroductionThis kit is specially designed for the efficient and rapid extraction of plasmids from 15-50 ml of bacterial fluids. On the basis of cell lysis by alkaline lysis method, it adopts unique silicon matrix membrane adsorption technology to bind plasmid DNA efficiently and exclusively, and each adsorption column can adsorb up to 250 µg of plasmid DNA; at the same time, it adopts a special buffer system and endotoxin removal filter to effectively remove endotoxin, genomic DNA, RNA, protein and other impurities. The plasmids obtained from this kit are of high purity and stable quality, and can be used for cell transfection, as well as DNA sequencing, PCR, in vitro transcription, endonuclease digestion and other experiments.Self-contained reagents: anhydrous ethanol, isopropanol.Pre-experiment Preparation and Important Notes1. All components are stable for 1 year in a dry, room temperature (15-30°C) environment, and longer by placing the adsorption columns at 2-8°C. Buffer P1 with RNase A is stable for 6 months at 2-8°C.2. Before the first use, add all of the RNase A solution to Buffer P1, mix well, and store at 2-8°C. Before use, it needs to be left at room temperature for a period of time, return to room temperature and then use.3. Anhydrous ethanol should be added to Buffer PW before the first use according to the instructions on the reagent bottle label.4. Please check Buffer P2 and Buffer E3 for crystallization or precipitation before use. If there is any crystallization or precipitation, the clarification can be restored by taking a water bath at 37℃ for a few minutes.5. Be careful not to touch Buffer P2 and Buffer E3 directly, and tighten the lid immediately after use.6. The amount and purity of extracted plasmid is related to the concentration of bacterial culture, strain type, plasmid size, plasmid copy number and other factors.7. The adsorption columns treated with Buffer PS should be used immediately to avoid leaving them for too long.Operation steps1.Take 15-50 ml of fresh bacterial solution from the overnight culture, add it to a centrifuge tube (self-prepared) and centrifuge at 5000 × g for 10 minutes to collect the bacteria, and aspirate all the supernatant as much as possible.2.Add 2.5 ml of Buffer P1 to the centrifuge tube in which the bacterial precipitate has been left (please check that RNase A has been added first) and suspend the bacterial precipitate by mixing thoroughly using a pipette or vortex shaker. Note: If the bacterial mass is not thoroughly mixed, it will affect the lysis effect and make the extraction amount and purity low.3.Add 2.5 ml of Buffer P2 to the centrifuge tube, mix gently up and down 8-10 times to fully lyse the organisms, and leave at room temperature for 3-5 minutes. At this point the solution should become clear and viscous. Note: Mix gently, do not shake vigorously, so as not to interrupt the genomic DNA and cause genomic DNA fragments to be mixed in the extracted plasmid. If the solution does not become clear, it suggests that the amount of bacteria may be too large and the lysis is not complete, and the amount of bacteria should be reduced.4.Add 2.5 ml of Buffer E3 to the centrifuge tube and mix immediately by turning up and down 8-10 times, at which time a white flocculent precipitate appears. Note: Buffer E3 should be mixed immediately after addition to avoid localized precipitation.5.Install the cap of the filter (Endo-Remover FX), transfer the solution obtained in step 4 to the filter, wait until the white flocculent precipitate floats on the upper layer of the solution, remove the cap of the filter, align the filter with a clean 15 ml centrifuge tube (supplied), and slowly push the handle (Plungers) to filter, so that as much as possible of the solution passes through, and the filtrate is collected in the centrifuge tube.6.Add 1/3 solution volume of isopropanol to the filtrate and mix upside down.7.Column Equilibrium: Add 1ml Buffer PS to the adsorption column (Spin Columns DX) that has been loaded into a 15ml centrifuge tube, centrifuge for 2 minutes at 2500 x g. Pour off the waste liquid from the centrifuge tube and put the adsorption column back into the centrifuge tube.8.The mixture of filtrate and isopropanol from step 6 was transferred to the equilibrated adsorption column (which had been loaded into a collection tube).9.Centrifuge at 2500 x g for 1 minute, pour off the waste solution in the collection tube and put the adsorption column back into the collection tube. Note: The maximum volume of the adsorption column is 4 ml, so the solution obtained in step 8 is passed through the column in 2 times.10.Add 2 ml of Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first), centrifuge at 2500 × g for 1 min, and pour off the waste liquid in the collection tube.11.Repeat step 10.12.The adsorbent column was put back into the collection tube and centrifuged at 2500 × g for 2 min, the waste liquid was poured off, and the column was left to dry at room temperature for 5 min.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can interfere with subsequent enzymatic reactions (digestion, PCR, etc.)13. Place the adsorption column in a new 15 ml centrifuge tube, add 0.5-1 ml Endo-Free Buffer EB to the middle of the adsorbent membrane, leave it at room temperature for 2-5 minutes, centrifuge it at 2500 × g for 2 minutes, and collect the plasmid solution into the centrifuge tube. -20°C to store the plasmid.Note: 1) In order to increase the recovery efficiency of the plasmid, the obtained solution can be reintroduced into the adsorption column, left at room temperature for 2-5 minutes, centrifuged at 2500 x g for 2 minutes, and the plasmid solution can be collected into a centrifuge tube.2) When the plasmid copy number is low or >10kb, Endo-Free Buffer EB can increase the extraction efficiency by preheating at 65-70°C in a water bath... Read More | 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 | Product content:M665754Component25 TStorageM665754ATris-HCl, 1 mM, PH 8.01 mL-20℃. Avoid freeze/thaw cycleM665754BE. coli Poly(A) Polymerase, 5 U/µL15 µL-20℃. Avoid freeze/thaw cycleM665754C10×Poly(A) Polymerase Buffer80 µL-20℃. Avoid freeze/thaw Product content:M665754Component25 TStorageM665754ATris-HCl, 1 mM, PH 8.01 mL-20℃. Avoid freeze/thaw cycleM665754BE. coli Poly(A) Polymerase, 5 U/µL15 µL-20℃. Avoid freeze/thaw cycleM665754C10×Poly(A) Polymerase Buffer80 µL-20℃. Avoid freeze/thaw cycleM665754DATP, 10 mM15 µL-20℃. Avoid freeze/thaw cycleM665754ERT Primer, 25 µM90 µL-20℃. Avoid freeze/thaw cycleM665754F5×SuperRT Buffer120 µL-20℃. Avoid freeze/thaw cycleM665754GUltraPure dNTP Mix, 10 mM each30 µL-20℃. Avoid freeze/thaw cycleM665754HSuperRT, 200 U/µL15 µL-20℃. Avoid freeze/thaw cycleM665754IRNase-Free Water1 mL-20℃. Avoid freeze/thaw cycle Product Introduction:This kit uses the method of adding a poly (A) tail at the 3 'end of miRNA to give miRNA a Poly (A) tail, followed by reverse transcription using Oligo (dT) - Universal tag universal reverse transcription primers to synthesize the first stranded cDNA corresponding to miRNA. The miRNA cDNA first strand synthesis kit contains all the reagents required for the miRNA 3 'end Poly (A) tail modification process and the reverse transcription process after modification. This kit has a very high Poly (A) modification and reverse transcription efficiency, which can range from 1 ng-2 µ The first strand of cDNA corresponding to miRNA was effectively obtained from the total RNA of g. And the operation is simple and fast, which can be used to simultaneously detect multiple miRNAs from a synthesized cDNA reaction. This not only reduces errors and saves samples, but also achieves high-throughput detection.Note: This kit must be used in conjunction with the miRNA fluorescence quantitative detection kit.Self prepared experimental materials: 1 ng-2 µ Total RNA of g, or 0.1 ng-1 µ Small molecule RNA of g.Notes:To prevent RNase pollution, attention should be paid to the following aspects:1. Use plastic products and gun heads without RNase to avoid cross contamination.2. Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use. Plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3. The solution should be prepared using water without RNase.4. Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.Usage:A. The process of miRNA adding Poly (A) tail:1.based on the amount of RNA used, dilute the total RNA of 10 mM ATP with 1 mM Tris (pH 8.0) according to the following formula: ATP dilution coefficient=5000/__ ngExample: If the initial amount of total RNA is 100 ng, then the ATP dilution coefficient is 5000/100=50. About to dilute ATP 50 times (1 µ 10 mM ATP plus 49 for l µ 1 mM Tris at pH 8.0.2. Add the following reagents to the pre cooled RNase free reaction tube in the ice bath to a total volume of 25 µ L. reagent 25 µlReaction system final concentration total RNA* X µl Up to 2 µg 10×Poly(A) Polymerase Buffer 2.5 µl 1× Diluted ATP in step "1" 1 µl / E. coli Poly(A) Polymerase, 5U/µl 0.5 µl 2.5 U RNase-Free Water up to 25 µl /*The total RNA used in the reaction must contain small molecule RNA.This process can also directly use small molecule RNA (recommended dosage of 2-5) µ L. Please determine the amount added based on the abundance of the target miRNA.3. Gently mix the above reaction solution and briefly centrifuge to collect the liquid at the bottom of the tube. Incubate at 37 ℃ for 15 minutes. After this process is completed, immediately proceed with the synthesis of the first strand cDNA or temporarily store it at -20 ℃. If long-term storage is required, it is recommended to store at -80 ℃.B. The process of synthesizing the first strand of modified miRNA cDNA:1. Add the reagents in the table below to the pre cooled RNase free reaction tube in the ice bath until the final volume reaches 20µl: reagent 20 µlReaction system The above Poly (A) reaction solution 4 µl UltraPure dNTP Mix ，10 mM each 1 µl RT Primer ，25 µM 3 µl 5×SuperRT Buffer 4 µl SuperRT ，200 U/µl 0.5 µl RNase-Free Water 7.5 µl2. Gently mix the above reaction solution and briefly centrifuge to collect the liquid at the bottom of the tube. Incubate at 42 ℃ for 50 minutes.3.85 ℃ for 5 minutes and terminate the reaction. The synthesized cDNA reaction solution can be directly used for fluorescence quantitative detection experiments or stored at -20 ℃ for future use... Read More | Products contentN665993Component240 TStorageN665993AIndex N501 Primers for Illumina240 µL-20℃. Avoid freeze/ Thaw cycle.N665993BIndex N925-N948 Primers for Illumina24×10 µL-20℃. Avoid freeze/ Thaw cycle. Products Introduction This kit is a companion kit to the transposase-Products contentN665993Component240 TStorageN665993AIndex N501 Primers for Illumina240 µL-20℃. Avoid freeze/ Thaw cycle.N665993BIndex N925-N948 Primers for Illumina24×10 µL-20℃. Avoid freeze/ Thaw cycle. Products Introduction This 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 IlluminaIndex N901-N996 Primer for Illumina... Read More |