| Description | DescriptionCAR10 is a kit that contains a selection of 10 carbohydrates/sugars: Arabinose, Fructose, Galactose, Glucose, α-Lactose, Maltose, Mannose, Ribose, Sucrose and Xylose, which may be used for general research, as reagents or as reference compounds in analytical procedures | B669951 Component 50T Storage B669951A Buffer ATL 15 mL RT B669951B Buffer AL 15 mL RT B669951C Buffer AW1 (concentrate) 13 mL RT B669951D Buffer AW2 (concentrate) 15 mL RT B669951E Buffer EB 15 mL RT B669951F Proteinase K 1.25 mL RT B669951G Spin Columns DM with Collection Tubes 50 sets B669951 Component 50T Storage B669951A Buffer ATL 15 mL RT B669951B Buffer AL 15 mL RT B669951C Buffer AW1 (concentrate) 13 mL RT B669951D Buffer AW2 (concentrate) 15 mL RT B669951E Buffer EB 15 mL RT B669951F Proteinase K 1.25 mL RT B669951G Spin Columns DM with Collection Tubes 50 sets RTProductsThis kit is suitable for extracting high purity total DNA from Gram-negative and Gram-positive bacteria. 106-108 cells can be processed at a time, and up to 20 µg of total DNA can be obtained within one hour without the need for toxic solvents such as phenol or chloroform, and without the need for ethanol precipitation. The optimized buffer system enables the DNA in the lysate to be efficiently and specifically bound to the silica matrix centrifugal adsorption column, while other contaminants can flow through the membrane, and the inhibitors of PCR and other enzymatic reactions can be effectively removed through a two-step washing step, and finally washed off with low-salt buffer or water, so that high-purity DNA can be obtained.The purified DNA can be used for downstream experiments such as digestion, PCR, Real-Time PCR, library construction, Southern Blot and molecular labeling, molecular labeling and other downstream experiments. Self-contained reagents: anhydrous ethanol; Enzymatic Lysis Buffer is required for extraction of Gram-positive bacteria.Enzymatic Lysis Buffer was prepared by 20 mM Tris, pH 8.0; 2 mM Na2-EDTA, pH 8.0; and 1.2% Triton X-100. 121°C sterilization for 20 minutes, and the appropriate amount of Lysozyme was added at a final concentration of 20 mg/ml. Pre-experiment Preparation and Important Notes1. Add 1.25ml 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 sample should be avoided, as this may result in smaller DNA fragments and a decrease in the amount of extracted DNA.3. If extracting genomes from bacterial cultures with high accumulation of secondary metabolites or thick cell walls, it is recommended that samples be collected early in the logarithmic phase.4. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the label of the reagent bottle before first use.5. Before use, please check Buffer GTL and Buffer GL for crystallization or precipitation. If crystallization or precipitation occurs, please re-dissolve Buffer GL and Buffer GTL in a 56℃ water bath.6. If the downstream experiments are sensitive to RNA contamination, 4µl of DNase-Free RNase A (100mg/ml) can be added before adding Buffer GL. RNase A is not provided in this kit.If the extracted samples are Gram-positive bacteria, customers need to prepare their own Enzymatic Lysis Buffer to treat the bacteria, which requires the use of Lysozyme (lysozyme) at a concentration of 20 mg/ml, which is not provided in this kit.Procedurei Extraction of genomic DNA from Gram-negative bacteria1. Take 1-5 ml of bacterial culture (106-108 cells, maximum 2×109 cells) and put it into a centrifuge tube (provided), centrifuge it at 12,000 rpm (~13,400×g) for 1 minute, and aspirate the supernatant as much as possible.2. Add 180 µl Buffer GTL to the precipitate and shake to resuspend the bacteria.3. Add 20 µl of Proteinase K, vortex and mix well, incubate at 56°C until the solution becomes clear, and invert or shake the centrifuge tube at intervals during the incubation to disperse the sample.Note: If RNA removal is required, add 4 µl of RNase A solution at a concentration of 100 mg/ml after the above steps are completed, shake to mix, and leave for 5-10 minutes at room temperature.4. Add 200µl Buffer GL and mix well with vortexing and shaking. Add 200µl of anhydrous ethanol and mix well with vortexing and shaking.Centrifuge briefly so that the solution on the walls of the tube collects at the bottom.Note: 1) If multiple samples are manipulated together, Buffer GL and anhydrous ethanol can be mixed in equal proportions and then added together, shaking to mix.2) The addition of Buffer GL and anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.5. Add all of the solution obtained in step 4 (including the precipitate formed) to the Spin Columns DM in the collection tube, or if the solution cannot be added all at once, transfer it several times. centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and return the column to the collection tube.6. Add 500 µl of Buffer GW1 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and return the adsorption column to the collection tube.7. Add 500 µl of Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.Note: Step 7 can be repeated if further DNA purity is required.8. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorbent column at room temperature for several minutes to dry thoroughly. Note: The purpose of this step is to remove residual ethanol from the adsorbent column; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).9. Place the adsorption column in a new centrifuge tube, add 50-200 µl Buffer GE to the middle part of the adsorption column overhanging the center of the adsorption column, leave it at room temperature for 2-5 minutes, centrifuge it at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20 ℃. note: 1) If the downstream experiments are sensitive to the pH or EDTA, the elution can be done with sterilized water. The pH of the elution solution has a great influence on the elution efficiency. If water is used as the elution solution it should be ensured that its pH is 7.0-8.5 (the pH of water can be adjusted to this range with NaOH), and the elution efficiency is not high when the pH is lower than 7.0.2) Incubation at room temperature for 5 minutes prior to centrifugation increases yield.3) Re-elution with an additional 50-200 µl Buffer GE or sterilized water can increase the yield.4) If the final concentration of DNA is to be increased, the DNA eluate obtained in step 9 can be re-spiked onto the adsorbent membrane and step 9 repeated; if the elution volume is less than 200 µl, the final concentration of DNA can be increased, but the total yield may be reduced. If the amount of DNA is less than 1 µg, elution with 50 µl Buffer GE or sterilized water is recommended.(5) DNA stored in water will be affected by acidic hydrolysis. For long-term storage, it is recommended to elute with Buffer GE and store at -20℃.i. Extraction of genomic DNA from Gram-positive bacteria1. Take 1-5 ml of bacterial culture (106-108 cells, maximum 2×109 cells) and put it into a centrifuge tube (provided), centrifuge it at 12,000 rpm (~13,400×g) for 1 minute, and aspirate the supernatant as much as possible.2. Add 180µl Enzymatic Lysis Buffer (self-provided) to resuspend the bacteria.Enzymatic Lysis Buffer is prepared as described in the Self-Prepared Reagents section in the front of the manual.3. Incubate at 37°C for 30 minutes.4. Add 20µl Proteinase K and mix well. Add 200µl of Buffer GL and mix well with vortexing and shaking.Note: Do not add Proteinase K directly to Buffer GL.Incubate at 5.56°C for 30 minutes.Note: 1) If desired, incubation at 95°C for 15 minutes will inactivate the pathogen, but 95°C incubation will cause some DNA degradation.(2) If RNA removal is required, add 4µl of RNase A solution at a concentration of 100mg/ml after the above steps are completed, shake and mix well, and leave for 5-10 minutes at room temperature.6. Add 200µl of anhydrous ethanol and mix well with vortex shaking.Note: The addition of anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.7. Add all of the solution obtained in step 6 (including the precipitate formed) to the Spin Columns DM that have been loaded into the collection tube, and if the solution cannot be added all at once, it can be transferred in several times. centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and put the column back into the collection tube.8. Add 500 µl of Buffer GW1 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.9. Add 500 µl Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge the column at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the column back into the collection tube.Note: Step 9 can be repeated if further DNA purity is required.10. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly.Note: The purpose of this step is to remove residual ethanol from the adsorption column; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).11. Place the adsorption column in a new centrifuge tube (self-provided), add 50-200 µl of Buffer GE to the center of the adsorption column overhanging the center of the adsorption column, let it stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20℃.Note: 1) If the downstream experiment is sensitive to pH or EDTA, you can use sterilized water for elution. The pH of the eluent has a great influence on the elution efficiency, if water is used as the eluent should ensure that its pH is 7.0-8.5 (you can use NaOH to adjust the pH of the water to this range), and the elution efficiency is not high when the pH is lower than 7.0.2) Incubation at room temperature for 5 minutes prior to centrifugation increases yield.3) Re-elution with an additional 50-200 µl Buffer GE or sterilized water can increase the yield.4) If the final concentration of DNA is to be increased, the DNA eluate obtained in step 11 can be re-spiked onto the adsorbent membrane and step 11 repeated; if the elution volume is less than 200 µl, the final concentration of DNA can be increased, but the total yield may be reduced. If the amount of DNA is less than 1 µg, elution with 50 µl Buffer GE or sterilized water is recommended.(5) DNA stored in water will be affected by acidic hydrolysis. For long-term storage, it is recommended to elute with Buffer GE and store at -20℃... Read More | Product introduction：Dualucif The firefly & Renilla assay kit (dual luciferase reporter assay kit) provides an effective means to detect the expression of genes. In DLR detection, the activities of firefly luciferase and Renilla luciferase can be detected in a single sample in turn. FirstProduct introduction：Dualucif The firefly & Renilla assay kit (dual luciferase reporter assay kit) provides an effective means to detect the expression of genes. In DLR detection, the activities of firefly luciferase and Renilla luciferase can be detected in a single sample in turn. First, luciferin was used as substrate to detect the activity of firefly luciferase, then substances inhibiting the catalysis of firefly luciferase were added, and coelenterazine was added to detect the activity of Renilla luciferase to achieve dual luciferase reporter gene detection. The bioluminescence system of luciferase and its substrate can detect gene expression very sensitively and efficiently. Usually, the transcriptional regulatory element or 5'promoter region of the gene of interest is cloned upstream of luciferase, or the 3'-utr region is cloned downstream of luciferase to construct a reporter gene plasmid, and then transfect the cells. After the cells are treated with appropriate drugs, the cells are lysed, and the transcriptional regulation effect of drug treatment on the target gene is judged by detecting the luciferase activity. Renilla luciferase is more often used as an internal reference for detecting transfection efficiency to eliminate the difference in cell number and transfection efficiency. Firefly luciferase is a protein with a molecular weight of about 61 kDa. 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. Renilla luciferase is a protein with a molecular weight of about 36 kDa. In the presence of oxygen, it can catalyze the oxidation of coelenteramide to coelenteramide, and also produce light signals in the process of coelenteramide oxidation. The optical signal of this kit can be measured by chemiluminescence instrument, microplate reader or liquid scintillation tester. The kit has the characteristics of rapid detection, high sensitivity, wide detection range and no interference of cell endogenous activity.Instruction：1.Cell lysis ( 1 ) Remove the medium and gently wash twice with PBS ( adherent cells can be operated directly, suspension cells need to be centrifuged to collect cells ). Add 1 × Lysis Buffer ( diluted component A with sterile water at 4 : 1 ) according to the following scheme, and then place the culture plate on a micro-oscillator at room temperature for 15 min to fully lyse the cells. Note : The pyrolysis products can be stored at room temperature for 6 h, and can be stored at − 70 °C for a long time ( the pyrolysis products cannot be repeatedly frozen and thawed ). ( 2 ) The pyrolysis products were centrifuged at 10000-15000 rpm for 3-5 min. After centrifugation, the supernatant was transferred into a new EP tube for subsequent detection. Note : Cells can be detected immediately after lysis, or frozen, and re-detected when needed. The frozen samples need to be thawed to room temperature for detection. 2. Preparation of working fluid ( 1 ) Restore all components to room temperature. ( 2 ) Dilute component C with component B to 0.2 mg / mL firefly luciferase working solution. Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the amount of a single experiment is small, it is recommended to be subpackaged into small specifications according to a single amount of use. ( 3 ) The E component was diluted into the renilla luciferase working solution with the D component, and the dilution method was 1 µL E component was added to the 49 µL D component. Note : Renilla luciferase working solution needs to be prepared now. 3.chemiluminescence value detection ( 1 ) According to the operation instructions of the instrument, the instrument with chemiluminescence detection function was opened, such as multifunctional microplate reader. The parameters were set, the determination time was 10 s, and the determination interval was 2 s. ( 2 ) each sample determination, take the sample 20-100 µL ( if the sample volume is enough, please add 100 µL ; if the sample amount is insufficient, the amount can be appropriately reduced, but the amount of detection holes needs to be consistent ). 1 × Lysis Buffer was blank control. ( 3 ) 100 µL firefly luciferase working solution was added to determine the RLU ( relative light unit ) value ( it is recommended that the microplate reader set up the Shaking mixing function ). Note : Since the luminescence is instantaneous, it is recommended to detect immediately after adding the firefly luciferase working solution. ( 4 ) 100 µL renilla luciferase working solution was added to determine the RLU ( relative light unit ) value ( Shaking mixing function is recommended for microplate reader ). ( 5 ) In the case of renilla luciferase as an internal reference, the RLU value measured by firefly luciferase was divided by the RLU value measured by renilla luciferase. According to the obtained ratio, the activation degree of the target reporter gene between different samples was compared. If firefly luciferase is used as an internal reference, similar calculations can also be performed.Component：Recommendation：It is recommended to use component B in advance to prepare 2 mg / mL storage solution, component B, component D and component C prepared as storage solution, and to carry out small batch packing according to the experimental requirements. All test working fluids are recommended to be used now to avoid repeated freezing and thawing.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. in order to obtain the best determination effect, when using a single tube chemiluminescence instrument for determination, the time from the mixing of sample and determination reagent to the pre determination should be controlled as much as possible; When using a multi-functional fluorescent microplate reader with chemiluminescence detection function, it is advisable to add all samples first, and then uniformly add firefly luciferase detection reagent. 3. the strongest wavelength of firefly luciferase catalyzed bioluminescence is 560 nm, and the strongest wavelength of Renilla luciferase catalyzed bioluminescence is 480 nm. 4. to prevent interference between holes, it is recommended to use white opaque orifice plate. 5. due to the influence of temperature on enzyme reaction, the sample and reagent should be measured after reaching room temperature. 6. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Study on gene expression regulation and promoter... Read More | Products contentN665954Component24 T96 TStorageN665954ATPS V136 µL144 µL-20℃. Avoid freeze/thaw cycle.N665954B5×FA Reaction Buffer96 µL384 µL-20℃. Avoid freeze/thaw cycle.N665954CTS Buffer72 µL288 µL-20℃. Avoid freeze/thaw cycle.N665954D2× Products contentN665954Component24 T96 TStorageN665954ATPS V136 µL144 µL-20℃. Avoid freeze/thaw cycle.N665954B5×FA Reaction Buffer96 µL384 µL-20℃. Avoid freeze/thaw cycle.N665954CTS Buffer72 µL288 µL-20℃. Avoid freeze/thaw cycle.N665954D2× PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw cycle. * This kit is suitable for human genomic DNA library construction, the starting template DNA input amount is 1 ng, our company also has 50 ng and 5 ng of human genomic DNA starting transposase method library construction kit, in order to get a higher quality library, different starting amount of DNA is recommended to use different kits. Products IntroductionThis kit is developed for Illumina's high-throughput sequencing platform and provides the enzyme premix system and reaction buffer for genomic DNA library construction, including all components except PCR primers. Compared with the traditional library construction kits, this kit adopts the new transposase method for library construction, which can complete DNA fragmentation, end repair and junction reaction in one simple enzymatic reaction, significantly reducing the amount of template, reducing the number of experimental steps, and shortening the time of library construction; it adopts the high-fidelity DNA polymerase for library enrichment, and the preference-free PCR amplification can expand the coverage area of the sequence, which can be used for efficient and effective sequencing. The use of high-fidelity DNA polymerase for library enrichment and preference-free PCR amplification broadens the coverage area of the sequence and enables efficient preparation of DNA libraries for Illumina's second-generation sequencing platform. The kit is suitable for use with 1 ng of starting template DNA, and all reagents in the kit have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction.Product Features● DNA fragmentation and junction ligation in one step.● Ultra-fidelity amplification minimizes amplification preference.Provide your own instruments, kits 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. Library PCR primer kit: It is recommended to use Kangwei transposase method for second generation sequencing multi-sample primer kit.4. Anhydrous ethanol, deionized water (pH between 7.0 and 8.0).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 Notes1. Avoid repeated freezing and thawing of reagents.2. PCR products are easily contaminated due to improper operation, resulting in inaccurate results. It is recommended to isolate the PCR reaction system preparation area from the PCR product purification area, and to use special pipettes to clean the experimental areas at regular intervals.3. Bead purification: the beads should be equilibrated to room temperature before use, all operations on the beads should be carried out at room temperature, 80% ethanol should be dispensed freshly, the beads should be rinsed and dried until the surface is free of liquid reflections and has a frosted appearance, insufficient drying of the beads will cause ethanol residue that will affect the subsequent experiments, and over-drying of the beads will affect the efficiency of DNA recovery.4. The kit is suitable for human genomic DNA library construction, if the DNA sample is a PCR product, it should be ensured that its length>.500 bp, since transposases do not work on DNA ends, it is recommended to extend the PCR product by 50-100 bp at each end of the PCR product to avoid low coverage of the ends for sequencing. Sample PreparationDNA purity requirement: A260/A280 = 1.8-2.0. Sample DNA: dissolved in ultrapure water.DNA quantification: Too much or too little DNA will affect the quality of the library. It is recommended to use Nano to test the purity of the genomic DNA and then use Qubit to test the concentration of the genome (do not use any absorbance-based assay for template quantification). Schematic diagram of DNA banking process procedureDNA fragmentation, junction reaction 1. Add the following reagents to a 200 µl PCR tube:2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:inactivation reactionAfter the DNA is fragmented, the enzyme is still in a high active state, so it should be removed from the PCR instrument immediately and terminated by adding the Reaction Termination Buffer, in order to prevent the DNA from being fragmented too much and resulting in smaller library fragments.1. Add 3 µl of TS Buffer to the PCR tube containing the fragmentation product.2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Incubate at room temperature for 5 min, or if the room temperature is too low, place the reaction on a PCR instrument at 25°C with the thermal cover closed.PCR amplification1. Add the following reagents to a 200 µl PCR tube.2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube. 3. Place the above PCR tubes in the PCR instrument with the thermal cap open, and the reaction program is as follows:Selective recovery of library DNA fragmentsIt is recommended to use CombiVision Magnetic Beads DNA Purification and Recovery Kit for selective recovery of DNA fragments. When different sizes of DNA fragments are required, the amount of magnetic beads used is different, please refer to the attached table for the specific amount of magnetic beads used.(If using other brands of magnetic beads, you need to figure out the optimal amount of magnetic beads by yourself).Note: Amplification products can also be fragment length sorted and purified using the Gum Recovery Kit. If there is no special requirement for library length distribution, amplification products can also be purified directly from DNA fragments without selective recovery of DNA fragments as described on page 4 of the manual.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Transfer the PCR products to a 1.5 ml centrifuge tube, rehydrate to 100 µl, add several volumes of magnetic beads equilibrated to room temperature, vortex for 5 seconds, and let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, and carefully aspirate the supernatant and transfer it to a new 1.5 ml centrifuge tube.Note: Do not discard the top clear.4. Add several volumes of magnetic beads to the supernatant, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.5. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA.Note: Do not discard the beads.6. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.7. Repeat step 6 once.8. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 20 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.9. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube. Table: Suggested amount of magnetic beads for different segment selection recoveryLibrary DNA fragment purificationWe recommend the use of the Kangwei Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. 50 µl of magnetic beads equilibrated to room temperature were added to the PCR product, vortexed and shaken for 5 seconds, and then left to stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 25 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube. Library quality controlDetermination of library concentrationIn order to obtain high-quality sequencing results, accurate quantification of DNA libraries is required, and the first recommendation is to use Real-timePCR methods are used for absolute quantification of DNA libraries. Additionally, fluorescent dye methods such as the Qubit method or the fluorescent dye picogreen method can be used; do not use quantification methods based on absorbance measurements here. The following approximate formula can be used to convert the molar concentration of the DNA library.Library fragment distributionThe prepared DNA libraries can be detected by agarose gel electrophoresis or Agilent 2100 Bioanalyzer.Range of segment length distributions... Read More | Product contentS666146Component50 T200 TStorageS666146ABuffer GR25 mL120 mLRTS666146BBuffer GL25 mL120 mLRTS666146CBuffer GW1 (concentrate)13 mL52 mLRTS666146DBuffer GW2 (concentrate)15 mL75 mLRTS666146EBuffer GE15 mL60 mLRTS666146FProteinase K1.25 mL4×1.25 mLRTS666146GSpin Columns DS with Product contentS666146Component50 T200 TStorageS666146ABuffer GR25 mL120 mLRTS666146BBuffer GL25 mL120 mLRTS666146CBuffer GW1 (concentrate)13 mL52 mLRTS666146DBuffer GW2 (concentrate)15 mL75 mLRTS666146EBuffer GE15 mL60 mLRTS666146FProteinase K1.25 mL4×1.25 mLRTS666146GSpin Columns DS with Collection Tubes50 sets 200 setsRTS666146HCentrifuge Tubes (1.5 mL)50 EA200 EARTProductsThis kit provides a simple and rapid method for the isolation and purification of total DNA from buccal swab samples. The kit adopts a silica matrix membrane that can specifically bind DNA and a unique buffer system to adsorb DNA efficiently and specifically, and 0.5-3.5 µg of genomic DNA can be obtained from each swab, and the extracted DNA fragments are large, pure and of stable and reliable quality. It is suitable for enzyme digestion, PCR, library construction, Southern hybridization and other experiments.Self-contained reagent: anhydrous ethanol.Pre-experiment Preparation and Important Notes1. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the label of the reagent bottle before first use.2. If precipitation is found in Buffer GL before use, dissolve Buffer GL in a 56°C water bath.3. All centrifugation steps can be performed at room temperature.4. Sampling: Use a buccal swab to wipe the inside of the mouth 6 times, dry for 2 hours and store. To ensure that the sample is not contaminated by food or drink, do not eat or drink for 30 minutes before sampling.Procedure1. The swab of the buccal swab was cut from the rod with scissors and placed in a 2mL centrifuge tube (supplied) and 400µL Buffer GR was added.Note: For genomic DNA without RNA contamination, add 4 µL of RNase A solution at a concentration of 100 mg/ml and shake to mix.2. Add 20 µL of Proteinase K and 400 µL of Buffer GL, immediately vortex and shake for 15 seconds and mix thoroughly.Note: Mix well immediately after adding Buffer GL; do not add Proteinase K directly to Buffer GL for use.3.56°C for 10 minutes and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.4. Add 400 µL of anhydrous ethanol, vortex and shake to mix thoroughly, and centrifuge briefly so that the solution on the wall of the tube collects at the bottom of the tube.Note: The addition of anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.5. Add the solution and precipitate obtained in the previous step to the Spin Columns DS in two batches of up to 700 µL at a time into the collection tube. centrifuge the column at 12,000 rpm (∼13,400 × g) for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.6. Add 500 µL of Buffer GW1 to the adsorbent column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorbent column back into the collection tube.7. Add 500 µL of Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge the column at 12,000 rpm for 3 minutes, pour off the waste liquid in the collection tube, and put the column back into the collection tube.Note: Step 7 can be repeated if further DNA purity is required.8. Centrifuge at 12,000 rpm for 1 minute and pour off the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly.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.).9. Place the adsorption column in a new 1.5 mL centrifuge tube, add 50 µL of Buffer GE or sterilized water to the middle of the adsorption column overhanging the column, let stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store at -20℃.Attention:(1) If the downstream experiment is sensitive to pH or EDTA, it can be eluted with sterilized water. The pH value of the eluent has a great influence on the elution efficiency. If the eluent is made of water, the pH value should be 7.0-8.5 (the pH value of water can be adjusted to this range by using NaOH), and the elution efficiency is not high when the pH value is lower than 7.0.2) For long-term storage, it is recommended to elute with Buffer GE and store at -20°C... Read More |