| Description | The miRNA extraction kit is specifically designed to isolate and purify miRNAs from various animal tissues, plant tissues, cells, serum, plasma and other samples. It can also extract small molecule RNAs such as siRNA and snRNA that are less than 200 nt, and can also be used for the extraction of The miRNA extraction kit is specifically designed to isolate and purify miRNAs from various animal tissues, plant tissues, cells, serum, plasma and other samples. It can also extract small molecule RNAs such as siRNA and snRNA that are less than 200 nt, and can also be used for the extraction of total RNA. This product combines phenol/guanidine lysis technology and silicon matrix membrane purification technology. The unique lysis solution can effectively inhibit RNases while removing most of DNA and proteins from cell or tissue samples through organic extraction. For some sensitive downstream experiments, if miRNA enrichment is required, this kit can be used to enrich miRNA separately. This product is suitable for a wide range of samples, with high purity of prepared RNA, and can be directly used for sensitive downstream applications, such as Northern Blot analysis, Real Time PCR, Microarray Analysis, etc. M665531Component50 TStorageM665531ATRIzon Reagent60 mL2-8℃. Protect from ligt.M665531BBuffer RWT (concentrate)15 mLRTM665531CBuffer RW2 (concentrate)11 mLRTM665531DRNase-Free Water10 mLRTM665531ESpin Columns RM with Collection Tubes50 setsRTM665531FSpin Columns RS with Collection Tubes50 setsRTM665531GRNase-Free Centrifuge Tubes (1.5 mL)50 EART Self prepared reagents: chloroform, anhydrous ethanol (newly opened or dedicated for RNA extraction).Preparation and important precautions before the experiment:To prevent RNase pollution, attention should be paid to the following aspects:1) Use RNase free plastic products and gun heads to avoid cross contamination.2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) Prepare the solution using water without RNase.4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.2. The extracted samples should avoid repeated freeze-thaw cycles, otherwise it will affect the quantity and quality of miRNA extraction.Before the first use, anhydrous ethanol should be added to Buffer RWT and Buffer RW2 according to the instructions on the reagent bottle label.4. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.Operation steps:Protocol A: miRNA enrichment (can be directly used for sensitive downstream experiments)1. Sample processing1a Organization: Grind the organization in liquid nitrogen. Add 1 ml of TRIzon Reagent to every 30-50 mg of tissue, shake and mix well. The sample volume shall not exceed one tenth of the volume of TRIzon Reagent.1b Single layer culture of cells: Remove the culture medium, add TRIzon Reagent, and add 1 ml of TRIzon Reagent every 10 cm2 (the amount of lysis solution depends on the area of the culture bottle).1c Cell suspension: Centrifuge to obtain cell precipitate, discard supernatant. Add 1 ml of TRIzon Reagent to every 5 x 106-1 x 107 cells (cells do not require washing).1d Plasma or serum: Take 200 µ Add 5 times the volume of TRIzon Reagent to plasma or serum samples, shake and mix well for 30 seconds.2. After adding TRIzon Reagent to the sample, blow it repeatedly several times to fully crack it. Leave at room temperature for 5 minutes to completely separate the protein nucleic acid complex.3. Optional steps: Centrifuge at 4 ℃ 12000 rpm (~13400 × g) for 5 minutes, take the supernatant, and transfer it to a new centrifuge tube (provided by oneself) (if the sample contains more proteins, fats, polysaccharides, etc., this step can be performed).4. Add chloroform to the supernatant and add 200 to every 1 ml of TRIzon Reagent used µ Chloroform, cover the tube, vigorously shake for 15 seconds, and let it sit at room temperature for 5 minutes.Centrifuge at 5.4 ℃ and 12000 rpm for 15 minutes. The sample is divided into three layers: red organic phase, middle layer, and colorless aqueous phase. Transfer the upper colorless aqueous phase to a new centrifuge tube (self prepared).6. Add 1/3 volume of anhydrous ethanol to the solution obtained in step 5, mix well, and transfer the obtained solution and precipitate together into the adsorption column RM (Spin Columns RM) that has been loaded into the collection tube. If you cannot add all the solution to the adsorption column at once, please transfer it multiple times. Centrifuge at 12000 rpm for 30 seconds, discard the adsorption column RM after centrifugation, and retain the effluent.7. Add 2/3 times the volume of anhydrous ethanol to the solution obtained in step 6 and mix well.8. Transfer the solution and precipitate obtained from the previous step into the adsorption column RS (Spin Columns RS) that has been loaded into the collection tube. If you cannot add all the solution to the adsorption column at once, please transfer it multiple times. Centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RS back into the collection tube.9. Add 700 to the adsorption column RS µ L Buffer RWT (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RS back into the collection tube.10. Add 500 to the adsorption column RS µ Buffer RW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RS back into the collection tube.11. Repeat step 10.12. Centrifuge at 12000 rpm for 1 minute and discard the waste liquid from the collection tube. Place the adsorption column RS at room temperature for a few minutes to thoroughly air dry. Note: The purpose of this step is to remove residual ethanol from the adsorption column RS, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).13. Place the adsorption column RS in a new RNase free centrifuge tube and add 30-50 to the middle of the adsorption column µ Place RNase Free Water at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store the obtained RNA solution at -70 ℃ to prevent degradation.Attention:1) The volume of RNase Free Water should not be less than 30 µ l. Small volume affects the recovery rate.2) If you want to increase RNA production, you can use 30-50 µ Repeat step 13 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column RS and repeat step 13Protocol B: Extraction of total RNA (including miRNA and other small molecule RNAs<200 nt), steps 1-5 are the same as protocol A.6. Add 1.25 times the volume of anhydrous ethanol to the solution obtained in step 5 and mix well.7. Transfer the solution and precipitate obtained from the previous step into the spin columns RM that have been loaded into the collection tube. If you cannot add all the solution to the adsorption column RM at once, please transfer it multiple times. Centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the collection tube.8. Add 700 to the adsorption column RM µ L Buffer RWT (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the collection tube.9. Add 500 to the adsorption column RM µ Buffer RW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the collection tube.10. Repeat step 9.11. Centrifuge at 12000 rpm for 1 minute and discard the waste liquid from the collection tube. Place the adsorption column RM at room temperature for a few minutes to thoroughly air dry. Attention: The purpose of this step is to remove residual ethanol from the adsorption column RM, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).12. Transfer the adsorption column RM into a new RNase free centrifuge tube and add 30-50 to the middle of the adsorption column µ Place RNase Free Water at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store the obtained RNA solution at -70 ℃ to prevent degradation. Attention: 1) The volume of RNase Free Water should not be less than 30 µ l. Small volume affects the recovery rate.2) If you want to increase RNA production, you can use 30-50 µ Repeat step 12 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column RM and repeat step 12... Read More | When apoptosis occurs, some DNA endonucleases will be activated. These endonucleases will cut off genomic DNA between nucleosomes and produce 180 bp-200 BP DNA fragments, which appear as a specific ladder pattern in agarose gel electrophoresis. When double strand or single strand breaks occur in When apoptosis occurs, some DNA endonucleases will be activated. These endonucleases will cut off genomic DNA between nucleosomes and produce 180 bp-200 BP DNA fragments, which appear as a specific ladder pattern in agarose gel electrophoresis. When double strand or single strand breaks occur in genomic DNA, a large number of sticky 3'-oh ends will be generated, which can interact with YF under the catalysis of deoxyribonucleotide terminal transferase (TDT) ®/ CY dUTP binding can directly detect apoptotic cells by fluorescence microscopy or flow cytometry. This kind of method is called terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Because normal or proliferating cells have almost no DNA breaks, there is no 3'-oh formation and they can rarely be stained. TUNEL method can stain intact single apoptotic nuclei or apoptotic bodies in situ, can accurately reflect the typical biochemical and morphological characteristics of apoptosis, and can detect a very small number of apoptotic cells, so it is widely used in the study of apoptosis. This kit has a wide range of applications and can be used to detect apoptosis in frozen or paraffin sections, as well as cultured adherent cells or suspended cells. It can selectively detect apoptotic cells, but not necrotic cells or cells with DNA strand breaks caused by irradiation and drug treatment. This kit detects cell apoptosis with a short time-consuming, one-step staining reaction and can be detected after washing.Composition: Composition 20T 50T A. aladdin®640 TUNEL Reaction Buffer 1 mL 2 ×1.25 mL B. TdT Enzyme 20 µL 50 µL C. Proteinase K (2 mg/mL) 40 µL 100 µL D. DNase I (2 U/µL) 5 µL 13 µL E. 10 ×DNase I Buffer 100 µL 260 µL Product parameters：642/662 nm； Instruction： Experimental materials (self provided)PBS buffer (1 x, pH~7.4). 0.2% Triton X -100 (PBS formulation). 0.1% Triton X -100 (PBS formulation, containing 5 mg/mLBSA)4% paraformaldehyde (prepared with PBS)Immunohistochemical penDewaxing solvent (paraffin section sample)Related reagents for paraffin section processingAnti fluorescence quenching and sealing agent. ddH2Oexperimental design. A. Positive control:Prepare positive control slides using DNaseI treatment. DNaseI can digest single or double stranded DNA and expose the 3 '- OH end, artificially causing cell apoptosis. One experiment per time is sufficient. (To verify if there are any issues with the experimental operation and reagent kit)B. Negative control:Use TUNEL Reaction Buffer without TdT Enzyme and replace TdT Enzyme with ddH2O. (Mainly to exclude non-specific staining caused by cell apoptosis, operational processes, and other reasons; and to adjust the exposure intensity of the shooting.)C. Experimental processing group.The experimental group operated normally according to the instructions.D. Experimental control group.The experimental group operated normally according to the instructions.Experimental steps1. Sample preparation:(1) For adherent cells or cell smearsa. Clean once with PBS.Note: If you are concerned that the cells on the cell smear may not adhere firmly, you can dry the sample to make the cells adhere more firmly.b. Fixation: Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and fix at 4 ℃ for 30 minutes. Clean twice with PBS.c. Translucency: Add an appropriate amount of 0.2% Triton X -100 (prepared with PBS) and let it penetrate at room temperature for 20 minutes. Clean twice with PBS.d. Step 2: TUNEL reaction.(2) For suspended cells or cell suspensionsa. Collect cells (3-5 x 106 cells), centrifuge at 1000 rpm for 5 minutes, and wash twice with PBS.b. Fixation: Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and resuspend the cells thoroughly. Fix at 4 ℃ for 30 minutes. Centrifuge at 2000 rpm for 5 minutes and clean twice with PBS.c. Translucency: Add an appropriate amount of 0.2% Triton X -100 (prepared with PBS) and let it penetrate at room temperature for 20 minutes. Centrifuge at 2000 rpm for 5 minutes and clean twice with PBS.d. Step 2: TUNEL reaction.(3) Paraffin tissue sectioninga. Dewaxing and hydration: Place the sliced samples sequentially in xylene I (10 min) → xylene II (10 min) → 100% ethanol I (5 min) → 100% ethanol II (5 min) → 95% ethanol (5 min) → 90% ethanol (5 min) → 80% ethanol (5 min) → 70% ethanol (5 min) → ddH2O rinse for 5 min, rinse twice.Note: Xylene is toxic and volatile. Please perform this operation in a fume hood.b. Use filter paper to dry the liquid around the sliced sample, and circle the sample contour with an immunohistochemical pen for downstream transparency and labeling.Note: If it is found that the contour circle of immunohistochemistry strokes is damaged in subsequent experimental operations, it needs to be redrawn in a timely manner.c. Transparency: Dilute 2 mg/mL of ProteinaseK solution with PBS in a ratio of 1:100 to a final concentration of 20 µ g/mL. Add 100 µ L dropwise to each sample to cover all sample areas. Incubate at 20-37 ℃ for 20 minutes.Note: Protein K can penetrate the cell membrane and nuclear membrane, allowing subsequent staining reagents to fully enter the nucleus for reaction and improve labeling efficiency. An excessively long incubation time increases the risk of tissue slices falling off the carrier film during subsequent washing steps, while a too short incubation time may result in insufficient permeability treatment and affect labeling efficiency. To obtain better results, the concentration, incubation time, and temperature of Protein K need to be optimized according to different types of tissue samples.d. Wash the slices twice with PBS, each time for 5 minutes. Use filter paper to remove excess liquid, and place the processed sample in a wet box to keep it moist.Note: Protein K must be washed thoroughly in this step, otherwise it will seriously interfere with subsequent labeling reactions.e. Step 2: TUNEL reaction.(4) Frozen tissue sectionsa. Fixation: Take out frozen sections and warm them back to room temperature. Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and fix at room temperature for 30 minutes. Wash twice with PBS for 10 minutes each time.Note: If you are concerned that formaldehyde cleaning may not be clean enough, it may affect the final dyeing effect. After formaldehyde fixation is completed, an appropriate amount of 2 mg/mL glycine can be added and washed for 10 minutes to neutralize the residual fixing solution, and then PBS cleaning can be carried out.b. Use filter paper to dry the liquid around the sliced sample, and circle the sample contour with an immunohistochemical pen for downstream transparency and labeling.Note: If it is found that the contour circle of immunohistochemistry strokes is damaged in subsequent experimental operations, it needs to be redrawn in a timely manner.c. Transparency: Dilute 2 mg/mL of ProteinaseK solution with PBS in a ratio of 1:100 to a final concentration of 20 µ g/mL. Add 100 µ L dropwise to each sample to cover all sample areas. Incubate at 20-37 ℃ for 20 minutes.Note: Protein K can penetrate the cell membrane and nuclear membrane, allowing subsequent staining reagents to fully enter the nucleus for reaction and improve labeling efficiency. An excessively long incubation time increases the risk of tissue slices falling off the carrier film during subsequent washing steps, while a too short incubation time may result in insufficient permeability treatment and affect labeling efficiency. To obtain better results, the concentration, incubation time, and temperature of Protein K need to be optimized according to different types of tissue samples.d. Wash the slices twice with PBS, each time for 5 minutes. Use filter paper to remove excess liquid, and place the processed sample in a wet box to keep it moist.Note: Protein K must be washed thoroughly in this step, otherwise it will seriously interfere with subsequent labeling reactions.e. Step 2: TUNEL reaction.(5) Positive treatment (only the positive control is subjected to this step, and other samples are directly subjected to the TUNEL reaction step)a. Dilute 10 x DNase I Buffer with ddH2O in a ratio of 1:10 to 1 x DNase I Buffer for later use.b. Drip 100 µ L of 1xDNase I Buffer onto the processed sample, covering all sample areas, and equilibrate at room temperature for 5 minutes.c. Dilute DNase I (2 U) with 1 x DNase I Buffer at a ratio of 1:100/ µ L) A working solution with a final concentration of 20 U/mL.d. Discard the buffer and add 100 µ Incubate DNase I working solution with a concentration of 20 U/mL at room temperature for 10 minutes.e. Discard DNase I working solution and clean twice with PBS.f. Step 2: TUNEL reaction.2. TUNEL reaction(1) Prepare TUNEL reaction solution (ready to use): / 1 sample 5 sample 10 sample TdT enzyme 1 µL 5 µL 10 µL YF®488/555/594/640 TUNEL Reaction Buffer 49 µL 245 µL 490 µL TUNEL Total volume of reaction solution 50 µL 250 µL 500 µL (2) For adherent cells, cell smears, or tissue sectionsa. Add 50 to each sample µ L TUNEL reaction solution, evenly cover the sample with the reaction solution. The appropriate time for dark incubation at 37 ℃ (recommended staining time for cells is 30 minutes to 1 hour, and tissue staining time is 2 hours).Note: 50 µ L TUNEL reaction solution is suitable for smear, slicing, or 96 well plates (other different well plates can adjust the volume of TUNEL reaction solution appropriately to cover cells). If the sample to be tested is a smear, slice, or in a 24 well plate, 12 well plate, or 6 well plate, anti evaporation film can be used, or self sealing bags or other appropriate materials can be used to cut circular plastic sheets slightly smaller than the holes. After adding TUNEL reaction solution dropwise, cover the sample to prevent the evaporation of TUNEL reaction solution and make the TUNEL reaction solution evenly cover the sample.b. Discard the TUNEL reaction solution, wash twice with PBS, and then wash three times with 0.1% Triton X -100 (PBS preparation, containing 5 mg/mL BSA) for 5 minutes each time. This way, free unreacted markers can be removed cleanly.c. (Optional) Add an appropriate concentration of 5 to each sample µ DAPI staining solution with a concentration of g/mL, incubated at room temperature in dark for 5 minutes. After staining, discard DAPI staining solution and wash twice with PBS for 5 minutes each time.d. (Optional) Slice sealing: Add 50 drops to each sample µ L anti fluorescence quenching sealing agent (anti fluorescence quenching sealing agent may not be suitable for certain dyes, it is recommended to conduct pre experimental testing for compatibility before the experiment), cover the cover glass, gently tap the cover glass with the blunt end of tweezers to remove bubbles and ensure complete sealing.e. Use filter paper to remove excess liquid and add 100 to the sample area µ Keep the sample moist with PBS and immediately observe under a fluorescence microscope.(3) For suspended cells or cell suspensionsa. Add 50 to each sample tube µ Gently resuspend cells in LTUNEL reaction solution and incubate at 37 ℃ in the dark for 30-1 hour. Gently resuspend cells with a micropipette every 15 minutes.b. Centrifuge at 2000 rpm for 5 minutes, discard TUNEL reaction solution, and wash twice with 0.1% Triton X -100 (PBS preparation, containing 5 mg/mLBSA) for 5 minutes each time. This way, free unreacted markers can be removed cleanly.c. Add 100 to each sample tube µ L concentration is 5 µ DAPI staining solution with a concentration of g/mL, incubated at room temperature in dark for 5 minutes.d. Join 400 µ L PBS resuspended cells and immediately detected with a flow cytometer or observed under a fluorescence microscope after smearing.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. when the staining background is heavy or non-specific staining is obvious, the staining time can be appropriately reduced. 3. it is recommended to add negative control and positive control groups during the experiment. 4. please wear mask and gloves when using component A. if it contacts the skin, please wash it with plenty of water immediately. 5. fluorescent dyes have quenching problems. Please try to avoid light to slow down fluorescence quenching. 6. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Late apoptosis detection, TUNEL Kit... Read More | 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 | Product content: G665990Component200 TStorageG665990ABuffer PG100 mLRTG665990BBuffer PS60 mLRTG665990CBuffer PW (concentrate)50 mLRTG665990DBuffer EB30 mLRTG665990ESpin Columns DM with Collection Tubes200 EART Product Introduction:This kit uses a new silicon-based plasma membrane technology and Product content: G665990Component200 TStorageG665990ABuffer PG100 mLRTG665990BBuffer PS60 mLRTG665990CBuffer PW (concentrate)50 mLRTG665990DBuffer EB30 mLRTG665990ESpin Columns DM with Collection Tubes200 EART Product Introduction:This kit uses a new silicon-based plasma membrane technology and reagent formulation. Through the unique centrifugal adsorption column and the DNA washing elution step, 100 bp-10 kb DNA fragments can be recovered and purified from ordinary or low melting point agarose gel. The sol speed is fast and the recovery rate is high. The sol solution contains a pH indicator, which can be used to determine whether the sol recovery has reached the optimal state based on its color. Each adsorption column can adsorb up to 10 µ G DNA, while effectively removing impurities such as primers, enzymes, mineral oil, and agarose. The purified and recovered DNA has high purity and concentration, good integrity, and can be directly used for molecular biology experiments such as sequencing, linking and transformation, labeling, and in vitro transcription.Self prepared reagents: anhydrous ethanol, isopropanol.Preparation and important precautions before the experiment:1.Before the first use, anhydrous ethanol should be added to the Buffer PW according to the instructions on the reagent bottle label.2. Before use, please check the Buffer PG. If crystallization or precipitation occurs, it can be left in a 37 ℃ water bath for 3-5 minutes to restore clarity.3. It is best to use a new electrophoresis buffer during electrophoresis to avoid affecting the electrophoresis and recovery efficiency; The following experiment requires high requirements, please use TAE electrophoresis buffer as much as possible.4.When cutting glue, the UV irradiation time should be as short as possible to avoid damage to DNA.5. The recovery rate is related to the initial amount of DNA and the elution volume. The smaller the initial amount, the smaller the elution volume, and the lower the recovery rate.6. Preheat the water bath to 50 ℃.7. Buffer PG contains a pH indicator. When the pH is ≤ 7.5, the color of the solution is yellow, and DNA can effectively bind to the membrane. When the pH is too high, the color of the solution turns orange red and purple, which needs to be adjusted.8. All centrifugation steps can be performed at room temperature.Operation steps:1. Cut the single purpose DNA strip from the agarose gel (try to cut the excess), put it into a clean centrifuge tube (self prepared), and weigh and calculate the weight of the gel (record the weight of the centrifuge tube in advance).Attention: If the volume of the adhesive block is too large, it can be cut into small pieces.2. Add one time of the volume of Buffer PG (if the gel weighs 100 mg, its volume can be regarded as 100 µ l. And so on.3.50 ℃ water bath and gently invert the centrifuge tube every 2-3 minutes until the sol turns yellow to ensure full dissolution of the gel block. If there are still unsolved glue blocks, you can add some more sol solution or continue to let it stand for a few minutes until the glue blocks are completely dissolved.Note: 1) After the gel is completely dissolved, the gel solution is yellow, and subsequent operations can be carried out; If the glue solution is orange red or purple, 10-30 can be added to the glue solution µ 3 M sodium acetate (pH 5.0), adjust the color of the solution to yellow before proceeding with subsequent operations.2) After the gel block is completely dissolved, it is best to lower the temperature of the gel solution to room temperature before loading the column. The adsorption column has a weaker ability to bind DNA at higher temperatures.4. (Optional step) When the recovered fragment is less than 300 bp, add 1/2 of the gel volume of isopropanol, and mix it upside down (if the gel weighs 100 mg, add 50 µ Isopropanol of L.5. Column balance: Add 200 to the spin columns DM that have been loaded into the collection tube µ Centrifuge at 13000 rpm (~16200 × g) for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.6. Add the solution obtained from steps 3 or 4 to the adsorption column that has been loaded into the collection tube, let it stand at room temperature for 2 minutes, centrifuge at 13000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.Attention: The volume of the adsorption column is 750 µ l. If the sample volume is greater than 750 µ L can be added in batches.7. Add 450 to the adsorption column µ LBuffer PW (please check if anhydrous ethanol has been added before use), centrifuge at 13000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.Note: If purified DNA is used for salt sensitive experiments (such as flat end ligation or direct sequencing), it is recommended to add Buffer PW and let it stand for 2-5 minutes before centrifugation.8. Repeat step 7.9.13000 rpm for 1 minute and discard the waste liquid from the collection tube.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.).10. Place the adsorption column into a new 1.5 ml centrifuge tube (provided by oneself), and add 50 drops to the middle position of the adsorption membrane in the air µ L Buffer EB, leave at room temperature for 2 minutes. Centrifuge at 13000 rpm for 1 minute and collect DNA solution- Store DNA at 20 ℃.Attention:1) To improve the recovery of DNA, the solution obtained by centrifugation can be re dropped onto the adsorption column, left at room temperature for 2 minutes, and centrifuged at 13000 rpm for 1 minute.2) The elution volume should not be less than 30 µ l. A small volume will affect the recovery efficiency.3) When recovering DNA fragments larger than 10 kb, Buffer EB should be preheated in a 50 ℃ water bath to increase recovery efficiency.Note: This reagent kit is also suitable for the purification and recovery of PCR products. Add an equal volume of Buffer PG to the PCR reaction solution and mix thoroughly (for small fragments with a recovery of less than 150bp, the solution volume can be increased to three times to improve the recovery rate). Follow step 5 above for further operations... Read More | Glycogen and starch generate glucose-1-phosphate (1PG/G1P) during the process of phosphohydrolysis. This reagent kit provides a simple, sensitive, and rapid determination method: Glucose-1-phosphate (1PG/G1P) is reduced from NADP+to NADPH by the sequential action of phosphoglucose mutase and Glycogen and starch generate glucose-1-phosphate (1PG/G1P) during the process of phosphohydrolysis. This reagent kit provides a simple, sensitive, and rapid determination method: Glucose-1-phosphate (1PG/G1P) is reduced from NADP+to NADPH by the sequential action of phosphoglucose mutase and phosphoglucose dehydrogenase. The content of glucose-1-phosphate (1PG/G1P) in the sample can be calculated by detecting the increase in NADPH at 340nm.Composition and preparation of reagent kit: Reagent name Specifications Save requirements Remarks Extraction solution Liquid 100mL x 1 bottle 4 ℃ storage / Reagent 1 Powder mg x 1 tube 4 ℃ storage Shake or centrifuge the reagent a few times before use to make it fall to the bottom, then dissolve it in 1.2mL of distilled water for later use. Reagent 2 Powder mg x 1 tube Store at -20 ℃ Shake or centrifuge the reagent a few times before use to make it fall to the bottom, then dissolve it in 1.2mL of distilled water for later use. Reagent 3 Liquid 16mL x 1 bottle 4 ℃ storage / Reagent 4 Powder mg x 1 tube Store at -20 ℃ Shake or centrifuge the reagent a few times before use to make it fall to the bottom, then add 1 Dissolve 1mL of distilled water for later use. TRC 1 powder 4 ℃ storage Only used to identify whether the reagents in the kit are normal (not involved in result calculation). Usage: Use a pre standard tube (GIP) to shake the powder a few times until it falls to the bottom, then add 0.5mL of distilled water and mix well to dissolveDilute GIP with a concentration of 4mg/mL and then dilute it four times to 1mg/mL for later use: follow the instructions in the sample addition table for the measuring tube operationRequired instruments and supplies:ELISA reader, 96 well plate, desktop centrifuge, adjustable pipette, mortar, ice and distilled water.Determination of glucose-1-phosphate (1PG/G1P) content:1. Sample preparation① Organizational sample:Suggest weighing around 0 1g of tissue, add 1mL of extraction solution, and homogenize in an ice bath. Centrifuge at 12000rpm, 4 ℃ for 10 minutes, take the supernatant, and place it on ice for testing.[Note]: If the sample size is increased, it can be extracted in a ratio of tissue mass (g) to extraction solution volume (mL) of 1:5-10.② Bacterial/cellular samples:Collect bacteria or cells into a centrifuge tube first, centrifuge and discard the supernatant; Take about 5 million bacteria or cells and add them to 1mLExtract solution, sonicate bacteria or cells (ice bath, power 200W, sonication for 3s, interval 10s, repeated 30 times); Centrifuge at 12000rpm at 4 ℃ for 10 minutes, take the supernatant, and place it on ice for testing.[Note]: If the sample size is increased, extraction can be carried out in a ratio of 500-1000:1 of bacteria/cell quantity (104) to extraction solution (mL).③ Liquid sample: direct detection.2. Machine testing:① Preheat the enzyme-linked immunosorbent assay (ELISA) reader for at least 30 minutes and adjust the wavelength to 340nm.② Thaw the reagent to room temperature (25 ℃);③ Add reagents to the 96 well plate in the following order according to the table:② Thaw the reagent to room temperature (25 ℃);③ Add reagents to the 96 well plate in the following order according to the table: Reagent name (µL） Measurement tube Blank tube (only done once) Reagent 1 10 10 Reagent 2 10 10 Reagent 3 150 170 Sample 20 / Mix well, incubate at room temperature (25 ℃) for 20 minutes, and then read A1 at 340nm (if the A value continues to increase, the incubation time needs to be extended until the absorbance value remains unchanged within 2 minutes). Reagent 4 10 10 Mix well, incubate at room temperature (25 ℃) for 20 minutes, and then read A2 at 340nm (if the A value continues to increase, the incubation time needs to be extended until the absorbance value remains unchanged within 2 minutes). Δ A=(A2-A1) measurement - (A2-A1) blank.[Note] 1 If the difference in Δ A is hovering around zero, the sample size V1 can be increased (such as increasing to 50 µ L, the three phases of the reagent should be reduced while keeping the total volume unchanged), or the sample sampling mass W can be increased. The changed V1 and W need to be substituted into the formula for recalculation.If the A2 value exceeds 1.2, the amount of sample added V1 can be reduced (such as to 10 µ L, the three-phase reagent should be increased while keeping the total volume unchanged), or the sample can be diluted with distilled water (keeping the sample addition system unchanged), and the changed V1 and D need to be substituted into the formula for recalculation.Result calculation:1. Calculated by sample weight:1PG/G1P content (µ g/g fresh weight)=[(Δ A ÷ (ε× d) × V2 × 106 × MR] ÷ (W × V1 ÷ V) × D=836 × Δ A ÷ W × D2. Calculated by the number of cells:1PG/G1P content (µ g/104 cell)=[(Δ A ÷ (ε× d) × V2 × 106 × MR] ÷ (500 × V1 ÷ V) × D=1.7 × Δ A × D. 3. Calculated by liquid volume:1PG/G1P content (µ g/mL)=[(Δ A ÷ (ε× d) × V2 × 106 × Mr] ÷ V1=836 × Δ A ε---NADPH Molar extinction coefficient，6.22×103 L/mol/cm； d---96 Orifice plate optical diameter，0.5cm； V---Add volume of extraction solution，1 mL； V1---Add sample volume，0.02mL V2---Total reaction volume；0.2mL=2×10-4L； W---Sample quality，g； Mr---Glucose-1-phosphate（1PG/G1P）Molecular weight；260； 500---Number of cells, in millions； D---Dilution ratio，Undiluted is 1。 /... Read More |