| Description | Product Introduction:At present, the conventional proteome sample pretreatment process has many problems, such as poor reproducibility, poor method stability, low sensitivity, being very time-consuming, and inability to be automated. To address the above issues, Aladdin has launched a new generationProduct Introduction:At present, the conventional proteome sample pretreatment process has many problems, such as poor reproducibility, poor method stability, low sensitivity, being very time-consuming, and inability to be automated. To address the above issues, Aladdin has launched a new generation of "All In One Tube" innovative products. This product is mainly designed for the processing of trace proteins (5-100µg). Even researchers without omics background can quickly prepare pretreated samples, which can be used for subsequent mass spectrometry analysis.Experimental Flowchart of Proteomics Pretreatment Kit (P1456469)Product Components and Storage Conditions:P1456469Component12 T24T48TStorageP1456469ALysis Buffer1.25 mL2.5 mL5 mL-20℃. Store in the dark.P1456469BDigest0.05 mL0.1 mL0.2 mL-20℃P1456469CBalance Buffer3 mL6 mL12 mL2-8℃P1456469DStop Buffer0.375 mL0.75 mL1.5 mLRTP1456469EWash Buffer I5 mL10 mL20 mLRTP1456469FWash Buffer II3.75 mL7.5 mL15 mLRTP1456469GWash Buffer III3.75 mL7.5 mL15 mLRTP1456469HElution Buffer6.25 mL12.5 mL25 mLRT. Store in the dark.P1456469ILoading Buffer0.25 mL0.5 mL1 mLRTP1456469JTip pillar12 T24T48TRTTrace proteins: Designed for the pretreatment of 5-100µg proteins.Simple operation: Enables rapid preparation of pretreated samples, requiring only a metal bath and a conventional centrifuge.High stability: Strict quality inspection for each batch ensures high reproducibility of experimental results.Operating Procedure:Lysis① Add 50µL of Lysis buffer to the EP tube containing the sample and mix by shaking.② Place the EP tube with Lysis buffer in a 95°C water bath for 10 minutes, then take it out and cool to room temperature.Enzymatic Digestion① Add 225µL of Balance buffer to the EP tube that has cooled to room temperature.② Add 3µL of Digest, mix well, and perform enzymatic digestion with shaking at 37°C and 1200rpm overnight (16 hours is recommended).Desalting (at room temperature)① Add 25µL of Stop buffer to the sample and vortex to mix.② Add 320µL of Wash buffer 1, shake vigorously for 3 minutes, centrifuge at 15000rpm for 3 minutes, and remove the supernatant.③ Transfer the lower layer sample into the Tip column, centrifuge at 2500rpm for 3-5 minutes until all liquid is centrifuged down. If the liquid flow rate is slow, the speed can be increased.④ Add 200µL of Wash buffer 2 (shake for 10-20 seconds before use) to the desalting column, centrifuge at 2500rpm for 3-5 minutes until all liquid is centrifuged down.⑤ Add 200µL of Wash buffer 3 to the desalting column, centrifuge at 2500rpm for 3-5 minutes until all liquid is centrifuged down.⑥ Put the desalting column into a new EP tube, add 200µL of Elution buffer to the desalting column, centrifuge at 2000rpm for 3-5 minutes until all liquid is centrifuged down.⑦ Repeat step ⑥, collect the eluates from both times, and freeze-dry them.⑧ Add 10µL of Loading buffer, vortex vigorously for 3 minutes, centrifuge at 20000g for 10 minutes, take an appropriate amount of sample, and then mass spectrometry detection can be performed. Taking the HF-X instrument as an example, 0.5-1µg of sample is sufficient for loading.Precautions:After aliquoting, Digest should be stored at -20°C.After aliquoting, Lysis Buffer should be stored at -20°C and avoid repeated freezing and thawing.This product is limited to scientific research use by professionals, and must not be used for clinical diagnosis or treatment, nor for food or drugs... Read More | 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 | Product introduction：Reporter gene detection is an important tool for analyzing the interaction between potential cis elements (such as promoters, enhancers and silencers) and trans acting factors in the flanking region of structural genes in the field of modern molecular biology. Firefly Product introduction：Reporter gene detection is an important tool for analyzing the interaction between potential cis elements (such as promoters, enhancers and silencers) and trans acting factors in the flanking region of structural genes in the field of modern molecular biology. Firefly luciferase is widely used in gene regulation and drug screening. Firefly luciferase is a protein with a molecular weight of about 61 KD. In the presence of ATP, magnesium ions and oxygen, it can catalyze the production of oxyluciferin from luciferin. In the process of luciferin oxidation, it will produce a light signal. The optical signal of this kit is a kind of instantaneous light, which needs to be detected immediately after adding the working solution. The half-life of optical signal is about 5 min.Instruction：1.Working fluid configuration ( 1 ) Restore all components to room temperature. ( 2 ) The component B ( stock solution ) was fully diluted with component A to prepare a 0.2 mg / mL firefly luciferase working solution, which was vortexed and shaken to ensure full mixing. Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the dosage of a single experiment is small, it is recommended to subpackage according to a single dosage. At room temperature, the activity decreased by about 10 % after the working solution was configured for 3 h, and the activity decreased by about 25 % after 5 h. 2.chemiluminescence value detection ( 1 ) The cell culture plate was taken out from the incubator and incubated at room temperature for 20 min to restore it to room temperature ( 22-25 ° C ). ( 2 ) Add the same volume of firefly luciferase working solution with the medium to the culture plate and mix well. ( 3 ) Incubation at room temperature for 5 min. Note : The incubation time can be adjusted according to cell type and cell number. ( 4 ) The values were read by multifunctional microplate reader or chemiluminescence instrument ( instrument parameters : the determination time was 10 s, the determination interval was 2 s ).Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. the strongest wavelength of bioluminescence catalyzed by firefly luciferase is 560 nm. 3. to prevent interference between holes, it is recommended to use white opaque orifice plate.Recommendation：Component B is recommended to use sterile water in advance to configure 2 mg / mL storage solution, A component and B component configured as storage solution, and small batch packaging according to the experimental requirements. The detection working fluid is recommended to be used now to avoid repeated freezing and thawing. Component：One-Step Firefly Luciferase Assay Buffer；D-Luciferin Scope of application：Mainly used for ADCC detection... 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