| Description | Inquire | Inquire | This product is a cDNA first strand synthesis kit specially prepared for the first step experiment of two-step RT-PCR. This product contains all the reagents required for reverse transcription from RNA templates to cDNA first strand, including HiFi MMLV reverse transcriptase, reaction buffer, This product is a cDNA first strand synthesis kit specially prepared for the first step experiment of two-step RT-PCR. This product contains all the reagents required for reverse transcription from RNA templates to cDNA first strand, including HiFi MMLV reverse transcriptase, reaction buffer, primers, dNTP, etc. The mutated HiFi MMLV reverse transcriptase RNase H activity is deficient, reducing RNA degradation in reverse transcription reactions and making it easier to obtain full-length cDNA. HiFi MMLV reverse transcriptase has strong thermal stability and can yield high yields of cDNA, making it simple and convenient to use. This system has high compatibility with subsequent PCR and quantitative PCR experiments, and is suitable for various DNA polymerase reactions. H665693 Component 100 T Storage H665693A HiFi-MMLV, 200 U/µL 100 µL -20℃. Avoid freeze/thaw cycle. H665693B 5×RT Buffer 500 µL -20℃. Avoid freeze/thaw cycle. H665693C Primer Mix 240 µL -20℃. Avoid freeze/thaw cycle. H665693D dNTP Mix, 2.5 mM Each 500 µL -20℃. Avoid freeze/thaw cycle. H665693E DTT, 0.1 M 240 µL -20℃. Avoid freeze/thaw cycle. H665693F RNase-Free Water 1 mL -20℃. Avoid freeze/thaw cycle. Product features:·RNase H -: Mutated HiFi MMLv reverse transcriptase with reduced RNase H activity, making it easier to obtain full-length cDNA.·Easy to use: The reagent kit contains all the reagents required for reverse transcription, except for RNA templates.Notes:1. During the operation process, RNase contamination should be avoided to prevent RNA degradation or cross contamination during experiments. It is recommended to perform RNA operations in specialized areas, use specialized instruments and consumables, and have operators wear masks and disposable gloves, and frequently change gloves.2. Disposable plastic containers should be used as much as possible for experiments. If glass containers are used, they should be treated with a 0.1% DEPC (diethyl pyrocarbonate) aqueous solution at 37 ℃ for 12 hours, and sterilized under high pressure at 120 ℃ for 30 minutes before use. Alternatively, glass containers should be sterilized under dry heat at 180 ℃ for 60 minutes before use. The sterile water used in the experiment should be treated with 0.1% DEPC and then subjected to high-pressure sterilization.3. All reagents in this reagent kit should be gently mixed upside down before use, avoiding foaming as much as possible, and used after brief centrifugation. The enzymes involved should be returned to -20 ℃ as soon as possible after use to avoid repeated freeze-thaw cycles.If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNAsin). This kit is not provided.Usage:Attention: 10 ng-5 µ G Total RNA can establish 20 µ Reaction system, if the total RNA content is greater than 5 µ g. Please expand the reaction system proportionallyi Steps for reverse transcription:1. Dissolve RNA templates, primers, dNTP Mix, DTT, RT Buffer, HiFi MMLV, and RNase Free Water and place on ice for later use.2. Prepare a reaction system according to the following table, with a total volume of 20 µ L. Reagent 20 µlReaction system Final concentration dNTP Mix，2.5 mM Each 4 µl 500 µM Each Primer Mix 2 µl / RNA Template X µl 1 ng-5 µg 5×RT Buffer 4 µl 1× DTT，0.1 M 2 µl 10 mM HiFi-MMLV，200 U/µl 1 µl / RNase-Free Water up to 20 µl / Attention:1) If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNAsin). This kit is not provided.2) Primer Mix is formulated from Oligo (dT) and Random Primer3. Vortex shake and mix well, briefly centrifuge to collect the solution on the pipe wall to the bottom of the pipe. 4. Incubate at 42 ℃ for 30-50 minutes and 85 ℃ for 5 minutes. After the reaction is complete, centrifuge briefly and cool on ice.5. Reverse transcripts can be directly used for PCR reactions and fluorescence quantitative PCR reactions, or stored at -20 ℃ for a long time.ii If the reverse transcription efficiency is low, or the RNA template secondary structure is complex and the GC content is high, the following steps are recommended:1. Dissolve RNA templates, primers, dNTP Mix, DTT, RT Buffer, HiFi MMLV, and RNase Free Water and place on ice for later use.2. Prepare the reaction system according to the following table, with a total volume of 13 µ L. Reagent 20 µlReaction system Final concentration dNTP Mix，2.5 mM Each 4 µl 500 µM Each Primer Mix 2 µl / RNA Template X µl 1 ng-5 µg RNase-Free Water up to 13 µl / 3. Incubate at 70 ℃ for 10 minutes and quickly ice bath for 2 minutes.4. Centrifuge briefly to collect the solution on the tube wall to the bottom of the tube.5. Continue to add the following reagents to the above reaction solution: Reagent 20 µlReaction system Final concentration 5×RT Buffer 4 µl 1× DTT，0.1 M 2 µl 10 mM HiFi-MMLV，200 U/µl 1 µl / Attention:1) If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNAsin). This kit is not provided.2) Primer Mix is formulated from Oligo (dT) and Random primer.6. Gently blow and mix well, incubate at 42 ℃ for 50 minutes, and incubate at 85 ℃ for 5 minutes.7. After the reaction is complete, centrifuge briefly and cool on ice.8. Reverse transcripts can be directly used for PCR reactions and fluorescence quantitative PCR reactions, or stored at -20 ℃ for a long time... Read More | This kit is used to extract miRNAs from various animal tissues, plant tissues and cells. The extracted miRNA molecule is complete and high purity, which is suitable for various molecular biology experiments such as Northern blot, real timepcr, miRNA microarray chip, in situ hybridization, This kit is used to extract miRNAs from various animal tissues, plant tissues and cells. The extracted miRNA molecule is complete and high purity, which is suitable for various molecular biology experiments such as Northern blot, real timepcr, miRNA microarray chip, in situ hybridization, RNase protection assay, etcComposition:Scope of application：Nucleic acid extraction and purificationInstruction：1.Experimental preparation：1.1.All reagents were prepared with DEPC-treated solvents. Please use RNase-free tip and centrifuge tube to avoid RNA degradation by RNase during extraction.1.2.70 % ethanol, -20C pre-cooling.2.Operational procedure：There is a slight difference in the operation of miRNA extraction from different samples. The specific steps are as follows :【 Extraction of miRNA from animal tissues】1.Take 20-40 mg tissue, transfer to a pre-cooled mortar, and add liquid nitrogen to grind into powder.Please click below to describe the amount of organization used :①RNA-rich tissue ( e.g. liver ) : no more than 30 mg②Tissues with low RNA content ( e.g., muscle ) : no more than 100 mg③When the amount of tissue used was less than 20 mg : the amount of R-I, R-II and isopropanol used was halved.④When the amount of tissue used was more than 40 mg : the use of R-I, R-II and isopropanol increased proportionally.2.Add 400 ul Buffer R-I, repeatedly aspirate 8-10 times with a syringe equipped with a 21-25 needle, and transfer to a 1.5 m : centrifuge tube ( provided in the kit ). 3.Add 150 µl BufferR-1l, swirl for 15-30 s, centrifuge at 12,000 X g for 5 min. [ Centrifugation at 4 °C is recommended ] 4.Take the supernatant to 1.5ml centrifuge tube, add 180 u anhydrous ethanol, mix evenly.5.The preparation tube was placed in a 2 m : centrifuge tube ( provided in the kit ), the mixture in step 4 was transferred to the preparation tube, and 12,000 X g was centrifuged for 1 min. [ 1 Centrifugation at 4 °C is recommended ; 2 miRNA in the filtrate, pay attention to preserve the filtrate. ]6.Abandon the preparation tube, add 500µl isopropanol to the filtrate, and mix evenly.7.12,000Xg centrifuged for 10 min, discard the supernatant.8.Add 700µl 70 % ethanol ( pre-cooled at -20 °C ), centrifuged at 12,000Xg for 5min.9.The supernatant was discarded and dried at room temperature for 5-10 min.10.70 ul Buffer TE ( nucdease-free ) or RNase-free water was added to the centrifuge tube to elute miRNA.【 Extraction of miRNA from plant tissue 】1.Take 30-150 mg tissue, transfer to a pre-cooled mortar, and add liquid nitrogen to grind into powder.Please click below to describe the amount of organization used :①Plant leaves : usually 10-80 mg② Plant fiber tissue : usually 100-150 mg③When the amount of plant leaf tissue was less than 30 mg : the amount of R-I, R-II and isopropyl alcohol used was halved.④When the amount of plant leaf tissue was more than 80 mg : the use of R-I, R-II and isopropanol increased proportionally.⑤When the amount of plant fiber tissue was more than 150 mg : the use of R-I, R-II and isopropanol increased proportionally.2.Add 400 ul BufferR-I, use a syringe with a 21-25 needle to repeatedly suck 8-10 times, and transfer to a 1.5mI centrifuge tube ( provided in the kit ). 3.Add 150 ul Buffer R-1I, vortex oscillation 15-30 s, 12.000 x g centrifugation 5 min. [ Centrifugation at 4 °C is recommended ]4.Take the supernatant to 1.5ml centrifuge tube, add 180 mountain anhydrous ethanol, mix evenly.The preparation tube was placed in a 2 mI centrifuge tube ( provided in the kit ), the mixture in step 4 was transferred to the preparation tube, and 12.000 xg was centrifuged for 1 min. It is recommended to centrifuge at 4 °C ; 2 miRNA in the filtrate, pay attention to preserve the filtrate. ]Abandon the preparation tube, add 500µl isopropanol to the filtrate, and mix evenly.7.12,000xg high heart for 10 min, discard the supernatant.8.Add 700 ul 70 % ethanol ( -20 °C precooling ), 12,000 xg centrifuge for 5 min.9.The supernatant was discarded and dried at room temperature for 5-10 min.10.70 ul Buffer TE ( nucdease-free ) or RNase-free water was added to the centrifuge tube to elute miRNA.【miRNA extraction from cells】Steps 1-3 According to the different ways of cell culture, two experimental methods, a or b, can be selected.a. Suspension cultured animal cells or cell suspension obtained from petri dishes or culture flasks or freshly isolated animal tissue single cell suspension :1a.Collect 2X 10 * -1X 10 ' cells, centrifuge 2,000Xg for 5 min, discard the supernatant ;2a. Add 400 µl Buffer R-I, repeatedly draw 8-10 times with a syringe containing 21-25 needles, and transfer to a 1.5 mI centrifuge tube ( provided in the kit ) ;3a. Add 150µl Buffer R1I, vortex oscillation 15-30s, 12.000Xg centrifugal 5min. [ build at 4 °C centrifugal ].b. Cells cultured on 96-well L, 24-well, 12-well or 6-well plates :Cells were collected from 96-well, 24-well, 12-well or 6-well culture plates, and the medium was discarded as much as possible, and 400 u / well Buffer R-I was added to each well, and the pipette gun was used to blow up and down 8-10 times ;2b.Transfer the above cell suspension to a 1.5ml centrifuge tube ( provided in the kit ), and repeatedly draw 8-10 times with a syringe containing 21-25 needles ;3b. Add 150 µl Bufflr R-II, swirl for 15-30 s, centrifuge for 5 min at 12,000 × g. [ Recommended at 4 °C ]4.Take the supernatant to 1.5ml centrifuge tube, add 180 mountain anhydrous ethanol, mixing evenly.5.The preparation tube was placed in a 2 ml centrifuge tube ( provided in the kit ), the mixture in step 4 was transferred to the preparation tube, and centrifuged at 12.000 Xg for 1 min. [ 1 Centrifugation at 4 °C is recommended ; 2 miRNA in the filtrate, pay attention to preserve the filtrate. ]6.Abandon the preparation tube, add 500 u of isopropanol to the filtrate, and mix evenly.7.12,000Xg high heart for 10 min, discard the supernatant.8.Add 700µ70 % ethanol ( pre-cooled at − 20 °C ), centrifuged at 12,000 × g for 5 min.9.Abandon the supernatant, dry at room temperature for 5 - 10 min.10.70 ul Bufer TE ( nucdease-free ) or RNase-free water was added to the centrifuge tube to elute mRNA.3.Flow chartMatters needing attention：Buffer R-I contains irritating compounds, when operating to wear latex gloves and glasses, to avoid contamination of the skin, eyes and clothes, be careful not to inhale the nose and mouth. If the skin, eyes, to immediately rinse with a lot of water or saline, if necessary, seek medical advice... Read More | Product contentS665868Component50 TStorageS665868ABuffer GL25 mLRTS665868BBuffer GW1 (concentrate)13 mLRTS665868CBuffer GW2 (concentrate)15 mLRTS665868DBuffer GE15 mLRTS665868EProteinase K2×1.25 mLRTS665868FSpin Columns DM with Collection Tubes50 setsRTProduct IntroductionThis kit is suitable Product contentS665868Component50 TStorageS665868ABuffer GL25 mLRTS665868BBuffer GW1 (concentrate)13 mLRTS665868CBuffer GW2 (concentrate)15 mLRTS665868DBuffer GE15 mLRTS665868EProteinase K2×1.25 mLRTS665868FSpin Columns DM with Collection Tubes50 setsRTProduct IntroductionThis kit is suitable for the extraction of genomic DNA from fresh saliva or saliva/preservation solution mixture.The purification process of this product does not require the use of toxic solvents such as phenol or chloroform, and ethanol precipitation is not necessary. The optimized buffer system enables DNA to bind heterogeneously to the silica matrix centrifugal adsorption column, and the inhibitors of PCR and other enzymatic reactions can be effectively removed by a two-step washing step, and finally eluted with a low-salt buffer or water to obtain high-purity DNA.The purified obtained can be directly used for enzyme digestion, PCR, Real-Time PCR, library construction, Southern Blot, molecular labeling and other downstream experiments.Self-contained reagent: anhydrous ethanolPre-experiment Preparation and Important Notes1. Repeated freezing and thawing of the sample should be avoided, as this may result in smaller fragments of extracted DNA and a decrease in the amount extracted.2. 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.3. Before use, please check whether Buffer GL appears to be crystallized or precipitated.Redissolve in a 56°C water bath.4. If the downstream experiments are sensitive to RNA contamination, 4 µL DNase-Free RNase A can be added in step 3(100 mg/mL).5. For prolonged storage of salivary DNA at room temperature, our Salivary DNA Preservation Tubes are recommended.Operation steps1. Add 400 µL of saliva sample or saliva/preservation solution mixture.Note: 1) Saliva mixtures added to the preservation solution require a 50°C water bath for 1 hour or an empty 50°C temperature chamber for 2 hours prior to extraction.2) If an increase in sample volume is required, multiply the volumes of Proteinase K, Buffer GL, and anhydrous ethanol in Steps 2-4, and the liquid can be transferred in multiple times in Step 5.2. Add 40 µL of Proteinase K.3. Add 400µL Buffer GL, vortex and shake to mix thoroughly, and water bath at 56℃ for 15-30 minutes.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, vortex for 15 seconds, and leave at room temperature for 2 minutes.4. Centrifuge briefly to remove water droplets from the inside of the tube cap. Add 400 µL of anhydrous ethanol and mix well by vortexing and shaking. Centrifuge briefly.Note: 1) Vortex and shake to mix immediately after adding Buffer GL and anhydrous ethanol.The addition of Buffer GL and anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.2) A sol-gel product may be formed after GL and anhydrous ethanol, in which case vigorous shaking or vortexing is recommended.3) The solution obtained in the previous step is added to the adsorption column in the Collection Tube.5. (Spin Column DM) in 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 (∼13,400 × g) for 1 min, pour off the waste solution in the collection tube, and put the adsorption column back into 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 put the adsorption 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 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 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 centrifuge tube (supplied), add 50-200 µL of Buffer GE or sterilized water to the middle of the adsorption column overhanging the column, let it stand at room temperature for 2-5 minutes, and centrifuge at 12,000 rpm for 1 minute to collect the DNA solution.-20°C to preserve DNA.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) Buffer GE preheated in a 65-70°C water bath and incubated at room temperature for 5 min before centrifugation can increase the yield.3) Because DNA preserved in water is subject to acidic hydrolysis, for long-term storage, elution with Buffer GE and storage at -20°C is recommended... Read More |