| Description | Inquire | Products content Box 1: Circularization reagentC666001Component16 TStorageC666001ASplint Oligo20 µL-20℃.Avoid freeze/thaw cycle. C666001B5×Splint Buffer T4250 µL-20℃.Avoid freeze/thaw cycle. C666001CDNA Ligase50 µL-20℃.Avoid freeze/thaw cycle. C666001DDigestion Products content Box 1: Circularization reagentC666001Component16 TStorageC666001ASplint Oligo20 µL-20℃.Avoid freeze/thaw cycle. C666001B5×Splint Buffer T4250 µL-20℃.Avoid freeze/thaw cycle. C666001CDNA Ligase50 µL-20℃.Avoid freeze/thaw cycle. C666001DDigestion Buffer20 µL-20℃.Avoid freeze/thaw cycle. C666001EDigestion Enzyme I70 µL-20℃.Avoid freeze/thaw cycle. C666001FDigestion Enzyme III25 µL-20℃.Avoid freeze/thaw cycle. Box 2: Magnetic Beads for DNA Purification and RecoveryC666001Component16 TStorageC666001GCMPure4×1.5 mL2-8℃Products IntroductionThe Cyclization Kit is a modular kit tailored for the MGI high-throughput sequencing platform. With this kit, PCR products after junction ligation can be prepared into single-stranded circular DNA libraries suitable for MGI sequencers. All reagents provided in the kit have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction. Provide your own instruments, reagents and consumables1. Magnetic frame: DynaMagTM-2 (Cat. No. 12321D) is recommended.2. "Qubit" 3.0 Fluorescence Quantimeter (ThermoFisher, Cat. No. Q33216)3. Qubit" ssDNA Assay Kit (Invitrogen, Cat. No. Q10212)4. Anhydrous ethanol, EB (10 mM Tris-HCl, pH 8.0), NF Water (pH between 7.0 and 8.0).5. reaction tubes: low adsorption PCR tubes with 1.5 mIEP tubes are recommended: 5.Tip: It is recommended to use a high quality filter tip to prevent contamination of kits and libraries. Pre-experiment Preparation and Important Notes 1. Sample preparation.PCR product: 2330 ng total (total amount when multiple PCR products are mixed) in a volume of 49 pL (if the volume of PCR product is insufficient, add NF Water to bring the total volume to 49 pl). -PCR product: Fragment size: The fragment peak is between 200-500 bp. -PCR product fragment size: Fragment peaks between 200-500 bp. -PCR product modification: Fixed sequences (with Index) for MGISEQ-2000, MGISEQ-200 and BGISEQ-500 sequencing platforms were added.2. Reagent preparation-Remove the corresponding reagents from the kit, centrifuge briefly, and place the enzyme mixture on ice until ready to use: buffers need to be dissolved at room temperature before use, then centrifuged with shaking and placed on ice until ready to use, and NF Water and EB are placed at room temperature until ready to use: "Please make up the mixture on ice:Precipitation may appear after the buffer in the kit is dissolved, the precipitation does not affect the function of the reagent, please shake and mix well until the precipitation disappears and then use. Schematic diagram of the cyclization process procedurecyclize 1. 1 wl of Splint Oligo was added to the 49JI PCR product. The product was denatured and incubated on a PCR instrument at 95°C for 3 min, then immediately transferred to an ice bath and allowed to stand for 2 min. 2. The reaction mixture was prepared on ice according to the following system. 3. Add 15ul of the above reaction mixture to 50µl of denatured DNA.4. Place the above PCR tubes on the PCR instrument under the following conditions Reaction. digest 1. Prepare the digestion reaction solution on ice according to the following system. 2. After the cyclization reaction, add 8l of digestion reaction solution directly to the cyclization system, mix well, centrifuge briefly and then place the PCR tube on the PCR instrument and react under the following conditions. 3. Purification was carried out immediately after the reaction.Purification of digestive products1. Remove CMPure at room temperature 30 minutes prior to use and mix well with shaking.2. Transfer the digested product to a 1.5 mIEP tube, pipette 340 pICMPure into the digested product, mix well by gently blowing 10 times with a pipette and incubate for 10 minutes at room temperature.3. Instantaneous centrifugation, place the EP tube on a magnetic rack and let stand for 5 minutes until the liquid is clear, pipette and discard the supernatant.4. Keep the EP tube fixed on a magnetic rack, add 250ul of freshly prepared 80% ethanol, let it stand at room temperature for 1 minute, then carefully discard the supernatant.5. Repeat step 4 once, try to suck up the liquid at the bottom of the tube: Note: Do not suck up the magnetic beads, so as not to affect the yield.6. Keep the EP tube fixed on the magnetic rack, open the cap and dry it at room temperature for 5-10 minutes.7. Remove the EP tube from the magnetic rack, add 35ul of EB or NF Water for DNA elution, pipette blow to mix and dissolve at room temperature for 10 min.8. Centrifuge instantaneously, place the EP tube on a magnetic rack and let stand for 2 minutes until the liquid is clarified, transfer the supernatant to a new EP tube. -Store at 20C and leave to prepare DNB... Read More | The Endo F Multi-Kit will deglycosylate N-linked glycans in both native and denatured conditions. Each enzyme has a distinct specificity for N-linked glycan release. One can choose to use the three enzymes in combination to completely remove all N-linked glycans present on a glycoprotein or peptide,The Endo F Multi-Kit will deglycosylate N-linked glycans in both native and denatured conditions. Each enzyme has a distinct specificity for N-linked glycan release. One can choose to use the three enzymes in combination to completely remove all N-linked glycans present on a glycoprotein or peptide, or to use each enzyme independently and thereby determine the type of N-glycans present.Product DescriptionThe Endo F Multi-kit is recommended to deglycosylate native proteins that are resistant to PNGase F cleavage under non-denatured conditions due to the glycan location within the protein’s three-dimensional structure, as these enzymes are known to be less sensitive to protein conformation.Each of the enzymes has a different N-linked glycan specificity:Endoglycosidase F1 cleaves high mannose and some hybrid type N-glycansEndoglycosidase F2 releases biantennary and high mannose glycans (at a 40X reduced rate)Endoglycosidase F3 will release triantennarry and fucosylated biantennary N-glycansContents1 vial: Endo F1- 20 µl (0.3 U)20 mM Tris-HCl pH 7.51 vial: Endo F2- 20 µl (0.1 U)10 mM sodium acetate, 25 mM NaCl, pH 4.51 vial: Endo F3- 20 µl (0.1 U)20 mM Tris-HCl pH 7.51 vial: 5x Reaction Buffer - 400 µl250 mM sodium acetate, pH4.51 vial: 5x Reaction Buffer - 400 µl250 mM sodium phosphate, pH5.5Specific ActivityDefined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micro-mole of denatured Ribonuclease B (Endo F1) or porcine fibrinogen peptides (Endo F2/F3) in 1 minute at 37°C, pH 5.5 (PH 4.5 for Endo F3). Cleavage is monitored by SDS-PAGE.FormulationThe enzymes are provided as a sterile-filtered solution.StabilitySeveral days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly.SpecificityEndo F1 cleaves Asparagine-linked (N-linked) high mannose or hybrid oligosaccharides. Endo F2 cleaves N-linked biantennary oligosaccharides and high mannose (at a 40X reduced rate). Endo F3 cleaves free or N-linked fucosylated biantennary or triantennary oligosaccharides,as well as triamannosylchitobiose core structures. These enzymes cleave between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. The recombinant version is not glycosylated, which may result in properties differing from the native protein.Quality & PurityEndo F1, Endo F2, and Endo F3 are tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The absence of exoglycosidase contaminants is confirmed by extended incubations with the corresponding pNP-glycosides. Directions for use 1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 34 µl final volume with de-ionized water. 2. Add 10 µl Endo F2 &F3 5x Reaction Buffer, 250 mM sodium acetate pH 4.5. Use Endo F1 buffer, 250 mM sodium phosphate pH 5.5 if you are using the Endo F1 enzyme alone. 4. Add 2.0 µl of each enzyme to the reaction. Incubate 3 hours at 37°C. Monitor cleavage by SDS-PAGE. Applications– Deglycosylation of native proteins resistant to PNGase F cleavage– Determination of glycan type (high mannose, biantennary, tri/tetrantennary)– Deglycosylating proteins which normally precipitate when deglycosylating– X-Ray CrystallographyThese three enzymes cleave asparagine-linked (N-linked) oligosaccharides between the two GlcNAc residues in the core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine, enhancing the solubility of the protein. In contrast, PNGase F removes the oligosaccharide intact... Read More | 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 |