| Description | Inquire | Calcein AM /PI Double Staining Kitis utilized for simultaneous fluorescence staining of viable and dead cells. This kit contains Calcein-AM and Propidium Iodide (PI) solutions, which stains viable and dead cells, respectively(Fig. 1). Calcein-AM, an acetoxymethyl ester of calcein, is highly Calcein AM /PI Double Staining Kitis utilized for simultaneous fluorescence staining of viable and dead cells. This kit contains Calcein-AM and Propidium Iodide (PI) solutions, which stains viable and dead cells, respectively(Fig. 1). Calcein-AM, an acetoxymethyl ester of calcein, is highly lipophilic and cell membrane permeable. Though Calcein-AM itself is not a fluorescent molecule, the calcein generated from Calcein-AM by esterase in a viable cell emits a strong green fluorescence (excitationat 490 nm, emission at515 nm). Therefore, Calcein-AM only stains viable cells. On the other hand, PI, a nuclei staining dye, cannot pass through a viable cell membrane. It reaches the nucleus by passing through disordered areas of dead cell membrane, and intercalates with the DNA double helix of the cell to emit red fluorescence (excitation: 535 nm,emmision: 617 nm). Since both calcein and PI-DNA can be excited with 490 nm, simultaneous monitoring of viable and dead cells is possible with a fluorescence microscope. With 545 nm excitation, only dead cells can be observed (Fig. 1). Since optimal staining conditions differ from cell line to cell line, we recommend that a suitable concentration of PI and Calcein-AM be individually determined. Please note that PI is suspected to be highly carcinogenic;careful handling is required.Required Equipment and Materials:Microscope with 490 nm excitation filter and 530 nm emission filter；CO2incubator；10 µl and 200 µl adjustable pipettes, PBSSolution A (Calcein-AM)；Solution B (PI) Storage Condition: -20oC ；Shipping Condition: blue ice.Application:Assay Procedure1)Add 2.5 µl Solution A and 12.5 µl Solution B to 5 ml PBS to prepare assay solution.*2)Wash the cell with PBS several times to remove residual esterase activity.3)Add 100uLof assay solution to200uL105~106CELLSsolution and incubate the mixture at 37oC for 15 min.4)Detect fluorescence using a fluorescence mircoscope with 490 nm excitationfor simultaneous monitoring of viable and dead cells.With 545 nm excitation, only dead cells can be observed.*The following steps may be necessary tooptimizethe suitable concentration of each reagent:1)Prepare dead cells by 10 min incubation in 0.1% saponin or 0.1-0.5% digitonin or by 30 min incubation in 70% ethanol.2)Stain dead cells with 0.1-10 µM PI solution to find a PI concentration that stains the nucleus only, not the cytosol.3)Stain dead cells with 0.1-10 µM Calcein-AM solution to find a Calcein-AM concentration that does not stain the cytosol. Then stainviable cells with that Calcein-AM solution to check whether the viable cell can be stained... Read More | 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 content： Component G665666 200 preps Buffer P1 60ml Buffer P2 60ml Buffer E3 60ml Buffer PW (concentrate) 25ml Buffer EB 30ml RNase A (10 mg/ml) 600 µl Spin Columns DM 200 with Collection Tubes 200Product Introduction:This reagent kit is suitable for extracting 1-5 ml of Product content： Component G665666 200 preps Buffer P1 60ml Buffer P2 60ml Buffer E3 60ml Buffer PW (concentrate) 25ml Buffer EB 30ml RNase A (10 mg/ml) 600 µl Spin Columns DM 200 with Collection Tubes 200Product Introduction:This reagent kit is suitable for extracting 1-5 ml of bacterial solution. On the basis of alkaline lysis of cells, it efficiently and specifically binds plasmid DNA through a new silicon-based membrane. Each adsorption column can adsorb up to 40% µ The plasmid DNA of g is effectively removed with a special buffer system to effectively remove impurities such as proteins. The yield and purity of plasmids obtained from this kit are high, and the quality is stable. It is suitable for downstream experiments such as cell transfection, DNA sequencing, PCR, PCR based mutations, in vitro transcription, transformed bacteria, and endonuclease digestion.Self prepared reagents: anhydrous ethanol, isopropanol.Preparation and important precautions before the experiment:1. All components can be stably stored for 1 year in a dry, room temperature (15-30 ℃) environment. The adsorption column can be stored for a longer time at 2-8 ℃. 2.Buffer P1 with RNase A added can be stably stored for 6 months at 2-8 ℃. Before use, add RNase A to Buffer P1 (add all RNase A provided in the reagent kit), mix well, and store at 2-8 ℃. Before use, it is necessary to leave it at room temperature for a period of time, and then use it after returning to room temperature.3.Before the first use, anhydrous ethanol should be added to the Buffer PW according to the instructions on the reagent bottle label.4. Before use, please check if there is any crystallization or precipitation in Buffer P2 and Buffer E3. If there is any crystallization or precipitation, you can take a water bath at 37 ℃ for a few minutes to restore clarity.5. Note that Buffer P2 and Buffer E3 contain irritating substances. Please wear gloves when operating and immediately cover the lid after use.6.The amount and purity of plasmid extraction are related to factors such as bacterial culture concentration, strain type, plasmid size, and plasmid copy number.7. The maximum volume of Spin Columns DM is 750 µ l. If the sample volume is greater than 750 µ L can be added in batches.Operation steps：1. Take 1-5 ml of overnight cultured bacterial solution and add it to a centrifuge tube (provided). Centrifuge at 13000 rpm (~16200 × g) for 1 minute to collect bacteria, and try to discard all the supernatant as much as possible.2. Add 200 to the centrifuge tube containing bacterial sediment µ Buffer P1 (please check if RNase A has been added first), mix thoroughly with a pipette or vortex oscillator, and suspend bacterial precipitation.Attention: If the bacterial blocks are not thoroughly mixed, it will affect the cracking effect, resulting in low extraction amount and purity.3. Add 200 to the centrifuge tube µ Buffer P2, gently invert and mix 8-10 times to fully lyse the bacterial cells. At this point, the solution should become clear and viscous.Attention: Mix gently and do not shake vigorously to avoid interrupting genomic DNA and mixing genomic DNA fragments in the extracted plasmid. If the solution does not become clear, it indicates that the bacterial count may be too large and the lysis may not be complete. The bacterial count should be reduced or the dosage of P1, P2, E3, and isopropanol should be increased proportionally.4. Add 200 to the centrifuge tube µ Buffer E3, immediately invert and mix 8-10 times, at which point white flocculent precipitates appear. Centrifuge at 13000 rpm for 5 minutes.Attention: After adding Buffer E3, it should be mixed evenly immediately to avoid local precipitation.5. Add 260 to the spin columns DM that have been loaded into the collection tube µ After adding isopropanol, immediately add the supernatant collected in step 4 and mix it upside down.Attention: After adding isopropanol, immediately add the supernatant and mix well to avoid isopropanol dripping into the collection tube after being left for a long time. The maximum volume of the adsorption column is 750 µ l. If the sample volume is greater than 750 µ l. Isopropanol and the supernatant can be collected in a centrifuge tube (provided by oneself), mixed well, and passed through the column in batches.6.13000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Add 400 to the adsorption column µ L Buffer PW (please check if anhydrous ethanol has been added first), centrifuge at 13000 rpm for 1 minute, and discard the waste liquid in the collection tube.8. Place the adsorption column in a new collection tube and add 50-100 to the middle of the adsorption membrane µ Centrifuge at 13000 rpm for 1 minute using buffer EB and collect the plasmid solution into a centrifuge tube- Store the plasmid at 20 ℃.Note: 1) To increase the efficiency of plasmid recovery, the obtained solution can be added back to the adsorption column, left at room temperature for 2-5 minutes, centrifuged at 13000 rpm for 2 minutes, and collected into a centrifuge tube.2) When the plasmid copy number is low or>10 kb, preheating the buffer EB in a water bath at 65-70 ℃ can increase the extraction efficiency... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export |