| Description | Inquire | Product Content D669986Component50 TStorageD669986ABuffer SA15 mLRTD669986B2×PCR MasterMix1 mL-20℃. Avoid freeze/thaw cycle.D669986CProteinase K12.5 mgRTD669986DProteinase K Storage Buffer1.25 mLRTProductsThis kit adopts a unique buffer system containing all the reagents for rapid Product Content D669986Component50 TStorageD669986ABuffer SA15 mLRTD669986B2×PCR MasterMix1 mL-20℃. Avoid freeze/thaw cycle.D669986CProteinase K12.5 mgRTD669986DProteinase K Storage Buffer1.25 mLRTProductsThis kit adopts a unique buffer system containing all the reagents for rapid preparation of genomic DNA and PCR amplification, and is suitable for one-step extraction of genomic DNA from various plant and animal tissues and bacteria and for PCR amplification. The whole extraction process does not require liquid nitrogen grinding, organic solvent extraction, anhydrous ethanol precipitation, and the quality of extracted DNA is stable. The 2×PCR MasterMix provided in this kit is a highly compatible PCR reagent that can amplify DNA samples efficiently and specifically, which includes DNA polymerase, dNTPs, MgCl2, reaction buffer, PCR reaction enhancer and so on. It is characterized by fast and easy, high sensitivity, high specificity, good stability, etc. It is especially suitable for high throughput screening.Pre-experiment Preparation and Important Notes1. Add the specified amount of Proteinase K Storage Buffer to Proteinase K to dissolve it and store it at -20℃. Do not leave the prepared Proteinase K at room temperature for a long time, and avoid repeated freezing and thawing to avoid affecting its activity.2. Repeated freezing and thawing of the samples should be avoided, as this will result in smaller DNA fragments and a decrease in the amount of extracted DNA.3. Before use, please check Buffer SA for crystallization or precipitation. If crystallization or precipitation occurs, please re-dissolve Buffer SA in a 56℃ water bath.4. The PCR MasterMix provided with this product is 2×, when using it, you need to add template and primer, and add RNase-Free Water to make up the volume, so that its concentration is 1× to carry out the reaction.Procedure1. Fetch:Plant material: take about 10 mg of sample in a centrifuge tube (provided); Animal material: take about 10 mg of sample in a centrifuge tube (provided);Bacteria: Take 200-800 µL of bacteria in good growth condition in a centrifuge tube (self-provided) and collect the bacteria.2. Add 200 µL of Buffer SA and vortex to mix.Note: In the case of plant leaves and animal tissues, they should be ground with a pestle and mortar as much as possible: in the case of plant seeds, they should be crushed and finely ground beforehand; bacterial and 1-3 mm rat-tail samples can be directly vortex lysed.3. Add 10µL of Proteinase K, mix well, incubate at 56℃ for 10 minutes, and treat at 95℃ for 5 minutes.Note: 1) In the case of animal tissue samples, the incubation time at 56°C may be extended to 30 minutes as appropriate; if there is any incompletely digested tissue, it should be removed as thoroughly as possible after centrifugation in the next step.2) Be careful not to exceed 5 minutes when treating at 95°C.4. 13,000 rpm (~17,900 x g), centrifugation for 5 minutes.5. Transfer the supernatant to a new centrifuge tube (self-prepared) and use it directly for PCR amplification, or store the solution at 4℃ or -20℃.6. PCR amplification:1) PCR reaction system:The following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.reagents20 µL systemfinal concentration2×PCR MasterMix10 µL1×Forward Primer, 10 µM1 µL0.4 µMReverse Primer, 10 µM1 µL0.4 µMTemplate DNA1-2 µL RNase-free Waterup to 20 µLNote: Please use the final concentration of 0.2-0.6µM as a reference for setting the range of primer concentration. If the amplification efficiency is not high, the concentration of primer can be increased; if a non-specific reaction occurs, the concentration of primer can be decreased, thus optimizing the reaction system.2）PCR reaction conditions:movetemptimingpremutability94°C2mindenaturation94°C30sannealing (metallurgy)55-65°C30s30-40 cyclesreach72°C60sultimate extension72°C5minNote: 1) In general, the annealing temperature is 5℃ lower than the melting temperature of the amplification primer Tm, and the annealing time is generally 30-60 seconds. When the desired amplification efficiency cannot be obtained, the annealing temperature should be lowered appropriately; when a non-specific reaction occurs, the annealing temperature should be raised, thus optimizing the reaction conditions.(2) The extension time is set according to the size of the fragment to be amplified, and the amplification efficiency of Taq DNA Polymerase included in this product is 1kb/30s. 3) The number of cycles can be set according to the downstream application of the amplification product. If the number of cycles is too low, the amplification is insufficient; if the number of cycles is high, the chance of mismatch will increase and the non-specific background will be serious. Therefore, the number of cycles should be minimized under the premise of ensuring the product yield.(3) Result detection: 5 µL of reaction product was taken at the end of the reaction and directly detected by agarose gel electrophoresis... Read More | Product content:E665636Component50 TStorageE665636ABuffer P115 mLRTE665636BBuffer P215 mLRTE665636CBuffer E315 mLRTE665636DBuffer PS15 mLRTE665636EBuffer PW (concentrate)10 mLRTE665636FEndo-Free Buffer EB10 mLRTE665636GRNase A (10 mg/mL)150 µLRTE665636HEndo-Remover FMwith Collection Tubes50 Product content:E665636Component50 TStorageE665636ABuffer P115 mLRTE665636BBuffer P215 mLRTE665636CBuffer E315 mLRTE665636DBuffer PS15 mLRTE665636EBuffer PW (concentrate)10 mLRTE665636FEndo-Free Buffer EB10 mLRTE665636GRNase A (10 mg/mL)150 µLRTE665636HEndo-Remover FMwith Collection Tubes50 EARTE665636ISpin Columns DMwith Collection Tubes50 EART Product Introduction:Endotoxins are a common pollutant in plasmid extraction. Due to the high sensitivity of eukaryotic cells to endotoxins, the presence of endotoxins in plasmids can greatly reduce the transfection efficiency of eukaryotic cells. This reagent kit provides a simple, fast, and efficient new method for extracting endotoxin free plasmids. The extracted plasmids remove endotoxins to the maximum extent possible and can effectively remove contamination of genomic DNA, RNA, proteins, etc. The operation is simple and convenient. This reagent kit is suitable for extracting 1-5mL 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 using a special buffer system and endotoxin removal filter column, effectively removing impurities such as endotoxins and proteins. The plasmid obtained from this kit has high purity and stable quality, making it particularly suitable for cell transfection. It can also be used for downstream experiments such as 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 ℃. Buffer P1 with RNase A added can be stably stored for 6 months at 2-8 ℃. 2. Before the first use, add all RNase A solution to Buffer P1, mix well, and store at 2-8 ℃. Before use, let it sit at room temperature for a period of time. After returning to room temperature, use.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. Be careful not to come into direct contact with Buffer P2 and Buffer E3, and immediately cover them tightly 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.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 30 seconds to collect bacteria, and try to discard all the supernatant as much as possible.2. Add 250 to the centrifuge tube containing bacterial sediment µ L 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 250 to the centrifuge tube µ L Buffer P2, gently invert and mix 8-10 times, allowing the bacterial cells to fully lyse. Leave at room temperature for 3-5 minutes. 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 high and the lysis may not be complete. The bacterial count should be reduced.4. Add 250 to the centrifuge tube µ L Buffer E3, immediately invert and mix 8-10 times until white flocculent precipitates appear. Let it stand at room temperature for 5 minutes. Centrifuge at 13000 rpm for 5 minutes, extract the supernatant, and add it to a filter column (Endo Remove FM). Centrifuge at 13000 rpm for 1 minute to filter, and collect the filtrate in a centrifuge tube (self provided).Attention: After adding Buffer E3, it should be mixed evenly immediately to avoid local precipitation. 5. Add 225 to the filtrate µ Mix L isopropanol upside down.6. Column balance: Add 200 to the spin columns DM that have been loaded into the collection tube µ L Buffer PS, centrifuge at 13000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Transfer the mixed solution of filtrate and isopropanol from step 5 to an equilibrium adsorption column (already loaded into a collection tube).8.13000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.Attention: The maximum volume of the adsorption column is 750 µ L. If the sample volume is greater than 750 µ L can be added in batches. 9. Add 750 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.10. Place the adsorption column back into the recovery manifold and centrifuge at 13000 rpm for 1 minute. 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.).11. Place the adsorption column in a new collection tube and add 50-100 to the middle of the adsorption membrane µ L Endo Free Buffer EB, let it stand at room temperature for 2-5 minutes, centrifuge at 13000 rpm for 2 minutes, 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 Endo Free Buffer EB in a water bath at 65-70 ℃ can increase the extraction efficiency... Read More | N665917 Component 1 mL 5 mL Storage N665917A 2×SYBR qPCR MasterMix 1 mL 5×1 mL -20℃. Avoid freeze/ Thaw cycle. N665917B qPCR Primer Mix 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917C DNA Standard A 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917 Component 1 mL 5 mL Storage N665917A 2×SYBR qPCR MasterMix 1 mL 5×1 mL -20℃. Avoid freeze/ Thaw cycle. N665917B qPCR Primer Mix 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917C DNA Standard A 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917D DNA Standard B 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917E DNA Standard C 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917F DNA Standard D 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917G DNA Standard E 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917H 50×High ROX 40 µL 200 µL -20℃. Avoid freeze/ Thaw cycle.Product IntroductionThis is a dye-based (SYBR Green I) qPCR NGS library quantification kit for cfDNA, which provides the reaction mixture, DNA primer mixture, standards, and sample dilutions required for the qPCR process, making it a complete reagent system that is easy and convenient to use. The fluorescent dye SYBR Green I contained in the reaction mixture binds to all double-stranded DNA. The kit uses a new chemically modified high-efficiency hot-start polymerase, the activation of the enzyme needs to be incubated at 95 ℃ for 10 min. the product is highly specific, high amplification efficiency, the length of the standard in the kit (about 270bp) is comparable to the average length of the cfDNA NGS libraries (250-300bp), which is able to quickly and accurately quantitate the concentration of the constructed cfDNA libraries. quantification.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration: Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96, etc.Instruments requiring Low ROX calibration: ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA7 System, Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and others.Instruments requiring High ROX calibration: ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, etc.NOTE: High Rox and Low Rox are formulated as described in Method of Use 2.Applicable scopeThis product is designed for the absolute quantification of the concentration of Illumina platform second generation sequencing libraries. The end of the library contains Illumina P5 and P7 microarray binding sequences, the length of which does not exceed 1kb, and the concentration is not less than 0.02pM can be used for quantitative experiments. The qPCR Primer Mix provided in the kit contains the following two primer sequences:Primer 1:5'-AAT GAT ACG GCG ACC ACC GA-3' Primer 2: 5'-CAA GCA GAA GAC GGC ATA CGA-3'The primer sequence can be used in advance to confirm whether the library can be amplified by that primer pair.UsageAmplification template preparationThe library samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.01-60 pM. 4°C on ice was set aside.qPCR reaction system preparationThe desired cryopreservation reagent is pre-melted completely and mixed by inverting several times before preparation, then centrifuged briefly and set aside.The base reaction system for 20 µl was as follows:Reagent20 µl Reaction system2×SYBR qPCR MasterMix10 µlqPCR Primer Mix0.8 µlTemplate4 µlddH₂O5.2 µlDescription: High Rox model: 1 µl High Rox per 50 µl of reaction system; Low Rox model: 1 µl High Rox per 500 µl of reaction system.Prepare a sufficient amount of reaction system mixture according to the need, mix well and add to the reaction wells in a volume of 16 µl per well, add the same volume of TE to the blank control, and then add the prepared standards and diluted samples to the corresponding reaction wells in a volume of 4 µl/well. It is recommended to use 20 µl reaction system, if you need to carry out a smaller system reaction, the system components can be reduced in equal proportion.3.qPCR reaction programIf the average length of the library is greater than 700bp, the annealing/extension time should be increased appropriately.Refer to the specific instrument setup program for dissolution curves.data analysisStandard curve productionThe standard curve was plotted according to the data processing Excel sheet. The correlation coefficient R2 of the standard curve should be not less than 0.99, and the slope should be located between -3.1 and -3.6 when the Ct value is the longitudinal coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment.DNA Standard NameDNA Standard ConcentrationDNA Standard A60 pMDNA Standard B6 pMDNA Standard C0.6 pMDNA Standard D0.06 pMDNA Standard E0.006 pMLibrary Concentration CalculationsThe difference in Ct between the three replicate wells of the experiment should be no more than 0.2, otherwise the invalid data should be deleted or the experiment should be repeated. Do not use the Ct outside the valid Ct range of the standard curve to calculate the concentration of the diluted libraries. Please refer to the data processing Excel of this product for the specific library concentration calculation method.matters needing attentionThese instructions should be read in detail before testing. It should be carried out by personnel with specialized experience or qualified by training.Mix gently by turning up and down, avoid foaming as much as possible, and centrifuge for a short time before use.Avoid repeated freezing and thawing of this product; repeated freezing and thawing may degrade product performance.When preparing reaction solutions, use new or non-contaminated tips and centrifuge tubes to prevent contamination as much as possible... Read More | Product contentP666142Component200 TStorageP666142ABuffer P160 mLRTP666142BBuffer P260 mLRTP666142CBuffer N380 mLRTP666142DBuffer PB35 mLRTP666142EBuffer PW (concentrate)25 mLRTP666142FBuffer EB30 mLRTP666142GRNase A (10 mg/mL)600 µLRTP666142HSpin Columns DM with Collection Tubes200 EART Product contentP666142Component200 TStorageP666142ABuffer P160 mLRTP666142BBuffer P260 mLRTP666142CBuffer N380 mLRTP666142DBuffer PB35 mLRTP666142EBuffer PW (concentrate)25 mLRTP666142FBuffer EB30 mLRTP666142GRNase A (10 mg/mL)600 µLRTP666142HSpin Columns DM with Collection Tubes200 EART Product IntroductionThis kit is suitable for extracting 1-5 ml of bacterial solution. Based on the lysis of cells by alkaline lysis method, it adopts a unique silica matrix membrane adsorption technology and reagent formulation, and efficiently and exclusively binds plasmid DNA in solution by centrifugal adsorption columns in a high-salt state, and each adsorption column can adsorb a maximum of 30 µg of plasmid DNA, and removes proteins, genomes, RNAs, and other impurities to the greatest extent possible. The plasmid DNA obtained can be directly used for cell transfection, PCR, digestion, sequencing, ligation and other biological experiments.Self-contained reagent: anhydrous ethanol.Pre-experiment Preparation and Important Notes1. All components can be stably stored in dry, room temperature (15-30℃) environment for 1 year, the adsorption column can be stored at 2-8℃ for a longer period of time, and Buffer P1 with RNase A can be stably stored at 2-8℃ for 6 months.2. Before the first use, add all the RNase A solution into Buffer P1, mix well, and store it at 2-8°C. Before use, leave it at room temperature for a period of time, and then use it after recovering to room temperature.3. Anhydrous ethanol should be added to Buffer PW according to the instructions on the label of the reagent bottle before first use.4. If precipitation is found in Buffer P2, Buffer N3, or Buffer PB before use, the clarification can be restored by water bath at 37℃ for a few minutes (please do not shake Buffer P2 violently).5. Be careful not to touch Buffer P2, Buffer N3 and Buffer PB 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.Procedure1. Take 1-5 ml of the overnight culture and add it to a centrifuge tube (self-prepared), centrifuge for 30 seconds at 13,000 rpm (~16,200×g) to collect the bacterial precipitate, and discard the supernatant as much as possible.2. Add 250 µl of Buffer P1 to the centrifuge tube with the bacterial precipitate (please check that RNase A has been added first), mix well using a pipette or vortex shaker, and suspend the bacterial precipitate.Note: If the bacterial mass is not thoroughly mixed, it will affect the lysis effect, resulting in low extraction and purity.3. Add 250µl of Buffer P2 to the centrifuge tube and mix gently up and down 4-6 times, mixing well to lyse the organisms, at which point the solution should become clear and viscous.Note: Mix gently, do not shake vigorously to avoid interrupting the genomic DNA and causing the extracted plasmid to be mixed with genomic DNA fragments. This step should take no more than 5 minutes to avoid damage to the plasmid.4. Add 350 µl of Buffer N3 to the centrifuge tube and immediately mix gently up and down for 8-10 times, mixing well so that a white flocculent precipitate should appear. centrifuge at 13,000 rpm for 5 minutes.Note: Buffer N3 should be mixed immediately after addition to avoid localized precipitation.5. Transfer the supernatant obtained in step 4 to the Spin Columns DM that have been loaded into the collection tube, centrifuge at 13,000 rpm for 30 seconds, pour off the waste liquid from the collection tube, and place the column back into the collection tube.6. Add 150 µl Buffer PB to the adsorption column and centrifuge at 13,000 rpm for 30 seconds.7. Add 400 µl Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first), centrifuge at 13,000 rpm for 1 minute, and pour off the waste liquid in the collection tube.8. Place the adsorbent column in a new centrifuge tube (supplied), add 50-100 µl Buffer EB to the middle of the adsorbent membrane, leave it at room temperature for 2 minutes, centrifuge at 13,000 rpm for 1 minute, and collect the plasmid solution into the centrifuge tube. -The plasmid solution was collected into the centrifuge tube.Note: 1) To increase the recovery efficiency of the plasmid, the resulting solution can be reintroduced into the adsorbent column, left at room temperature for 2 minutes, centrifuged at 13,000 rpm for 1 minute, and the plasmid solution collected into a centrifuge tube.2) For low plasmid copy number or >10 kb, Buffer EB is preheated at 65-70°C in a water bath to increase extraction efficiency... Read More |