| Description | EnzymoPure™II M-MLV Reverse Transcriptase (RNase H-) produced by aladdin is an optimized Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase without ribonuclease H (RNase H) activity. It is a DNA polymerase that uses single stranded RNA or DNA as template to synthesize complementary EnzymoPure™II M-MLV Reverse Transcriptase (RNase H-) produced by aladdin is an optimized Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase without ribonuclease H (RNase H) activity. It is a DNA polymerase that uses single stranded RNA or DNA as template to synthesize complementary DNA strands in the presence of primers. It is one of the most widely used reverse transcriptase.sApplication：First strand cDNA synthesis using total RNA or mRNA as template; DNA probe labeling with fluorescence, biotin, digoxin or isotope; RNA analysis by primer extension.This product is highly cost-effective. The RT II M-MLV reverse transcriptase (RNase H-) is a recombinant reverse transcriptase expressed and purified from E. coli transformed with the expression plasmids carrying the pol gene fragment of M-MLV with RNase H coding region removed. The enzyme has been engineered to have better thermal stability and higher reverse transcription activity.Definition of enzyme activity: One unit of the enzyme incorporates 1nmol of dTTP into acid-precipitable material in 10 min at 37℃ using poly(A)•oligo(dT)12-18 as template-primer. The reaction system contains 50mM Tris-HCl (pH8.3), 75mM KCl, 3mM MgCl2, 10mM DTT, 0.5mM [3H]-dTTP and 0.4 mM poly(A)•oligo(dT)12-18.Purity: Free from DNA endonuclease, DNA exonuclease, phosphoesterase and RNase.Enzyme storage buffer: 20mM Tris-HCl (pH7.5), 300mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01%(v/v) NP-40 and 50% (v/v) glycerol.Inactivation or inhibition: RT II M-MLV Reverse Transcriptase (RNase H-) can be inactivated by incubation at 80ºC for 10 minutes, or inhibited by EDTA, EGTA, inorganic phosphates, pyrophosphates and polyamine.This product has high reverse transcription activity, and is able to synthesize long cDNA. The RT II M-MLV Reverse Transcriptase (RNase H-) lacks RNase H activity, and does not degrade the RNA strand of an RNA-DNA hybrid, thus facilitating the synthesis of long cDNA. It can easily synthesize cDNA up to 8kb (Figure 1). The maximum length of synthesized cDNA by this product can exceed 10kb. Figure 1. Agarose gel electrophoresis of PCR products amplified from the cDNA template that was synthesized by the RT II M-MLV reverse transcriptase (RNase H-) (, #). cDNA ranging from 0.2kb to 8kb in length can be synthesized high efficiently.This product exhibits high thermal stability. This product has an optimum working temperature of 42-45ºC, but it is still highly active at 50ºC (Figure 2). Figure 2. Agarose gel electrophoresis of PCR products amplified from the cDNA templates that were synthesized by the RT II M-MLV reverse transcriptase (RNase H-) (, #) and by the M-MLV reverse transcriptase (RNase H-) (Beytome, ), respectively. 500ng of total RNA extracted from NIH3T3 cells was reverse transcribed by reverse transcriptase in a reaction volume of 20µl at difference temperatures as indicated in the figure. One microliter of each reverse transcription reaction product was taken for PCR amplification.The concentration of this product is 200U/µl. When 20µl of reverse transcription reaction volume is used, the S, M and L are sufficient for 50, 250 and 1000 reactions, respectively.Enzyme storage buffer：20mM Tris-HCl (pH7.5), 300mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01%(v/v) NP-40 and 50% (v/v) glycerol.Inactivation or inhibition：RT II M-MLV Reverse Transcriptase (RNase H-) can be inactivated by incubation at 80ºC for 10 minutes, or inhibited by EDTA, EGTA, inorganic phosphates, pyrophosphates and polyamine.This product has high reverse transcription activity, and is able to synthesize long cDNA. The RT II M-MLV Reverse Transcriptase (RNase H-) lacks RNase H activity, and does not degrade the RNA strand of an RNA-DNA hybrid, thus facilitating the synthesis of long cDNA. It can easily synthesize cDNA up to 8kb (Figure 1). The maximum length of synthesized cDNA by this product can exceed 10kb. Figure 1. Agarose gel electrophoresis of PCR products amplified from the cDNA template that was synthesized by the RT II M-MLV reverse transcriptase (RNase H-). cDNA ranging from 0.2kb to 8kb in length can be synthesized high efficiently.This product exhibits high thermal stability. This product has an optimum working temperature of 42-45ºC, but it is still highly active at 50ºC (Figure 2). Figure 2. Agarose gel electrophoresis of PCR products amplified from the cDNA templates that were synthesized by the RT II M-MLV reverse transcriptase (RNase H-) and by the M-MLV reverse transcriptase (RNase H-) (Beytome, D7159), respectively. 500ng of total RNA extracted from NIH3T3 cells was reverse transcribed by reverse transcriptase in a reaction volume of 20µl at difference temperatures as indicated in the figure. One microliter of each reverse transcription reaction product was taken for PCR amplification.The concentration of this product is 200U/µl. When 20µl of reverse transcription reaction volume is used, the D7160S, D7160M and D7160L are sufficient for 50, 250 and 1000 reactions, respectively.Precautions：Please refer to the instructions for reverse transcription of RNAs with high GC content.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear lab coat and disposable gloves during the operation.Instructions for Use：1. First-strand cDNA synthesis.a. Set up the first-strand cDNA synthesis reaction in a nuclease-free PCR tube on ice or at room temperature as follows. RNase Inhibitor and dNTP mix can be purchased from. Template (one of the three types of RNA)Total RNA0.1ng-5µgPoly(A) RNA/mRNA10pg-0.5µgSpecific RNA0.01pg-0.5µgPrimer (one of the three types of primer)Oligo(dT)18 Primer0.5µg (or 100pmol)Random Hexamer Primer0.2µg (or 100pmol)Gene-specific Primer15-25pmol(Optional) For RNAs with high GC content (e.g. ＞55%) or complex secondary structures, incubate the mixture of primer and template at 65ºC for 5 minutes, and immediately put it on ice to disrupt RNA secondary structures.DEPC-treated Water-To 13.7µl*Reaction Buffer (5X)-4µlRNase Inhibitor (40U/µl)-0.5µldNTP Mix (25mM each)-0.8µl**RT II M-MLV Reverse Transcriptase (RNase H-)-1µl***Total Volume-20µl* ‘To 13.7µl’ means filling the mixture of template and primer to a total volume of 13.7µl with DEPC-treated water.** The volume of dNTP mix varies depending on the concentration of dNTP stock. If the volume of dNTP is not 0.8µl, adjust the volume of DEPC-treated water accordingly.*** If gene specific primers or Oligo(dT)18 primers are used to reverse transcribe the cDNA over 5kb, the volume of RT II M-MLV reverse transcriptase (RNase H-) should be increased to 2µl.b. Mix the reaction by vortex or pipetting gently, centrifuge briefly to allow liquid to accumulate at the bottom of PCR tube.c. Incubate the reaction at 42ºC for 60min if Oligo(dT)18 primer or gene-specific primer is used. If random hexamer is used, carry out incubation at 25℃ for 10min, followed by incubation at 42℃ for 60min. Note: For RNA template with high GC content or secondary structures, incubate the reaction at 50℃ for 60min in order to improve the reverse transcription efficiency.d. Stop the reverse transcription by incubating the reaction at 80ºC for 10min to inactivate RT II M-MLV reverse transcriptase (RNase H-). Note: Heat-inactivation of reverse transcriptase is not recommended for long cDNA over 5kb, as this method may cause shearing of long cDNA fragments. In such a case, phenol-chloroform extraction or column purification can be considered.e. The reverse transcription products can be used directly for subsequent experiments such as PCR, or stored at -20℃ for future use. We recommend using 2µl reverse transcription products in a PCR reaction volume of 50µl.2. For other applications such as primer extension and probe labeling, please refer to reference related to M-MuLV reverse transcriptase (RNase H-).FAQ：1. The reverse transcription product of total RNA is invisible after electrophoresis.It is a normal phenomena, because the amount of RNA template is low, and the amount of reverse transcription products in different size is even lower. 2. No specific product can be amplified from the reverse transcription product.a. To exclude the problem of PCR reaction system or reverse transcription product, use gene-specific primers to amplify internal reference genes, such as actin and GAPDH. If reference genes can be amplified but not the target gene, it indicates primers of target gene are not well designed, the expression of the target gene is too low to be detected, or the reverse transcription efficiency is low. b. Template RNA may be degraded. The integrity of total RNA can be checked by agarose gel or on-chip electrophoresis. Intact total RNA exhibits sharp, clear 28S and 18S rRNA bands, and the 28S rRNA band should be approximately twice as intense as the the 18S rRNA band. A ratio less than 2 indicates the degradation of total RNA and new total RNA should be prepared. c. RNA samples may contain some components that inhibit the activity of reverse transcriptase. Those contaminants include phenol, SDS, EDTA, guanidine salts, phosphoric acid, pyrophosphoric acid, polyamine, and spermidine etc, which can be removed effectively by column purification or precipitation, washing, and redissolution. Total RNA extracted by Zol (, #R0011) or Trizol (, #R0016) usually can meet the requirements of reverse transcription.d. Insufficient templates for reverse transcription. When DNase I used to remove the residual DNA in RNA sample is subjected to heat-inactivation prior to reverse transcription, EDTA should be added to RNA sample at a final concentration of 2.5mM to protect RNA from degradation under high temperature. Additionally, to amplify a specific gene, it is necessary to extract RNA from tissues in which the target gene is highly expressed. e. Inappropriate primer is used for reverse transcription. Random hexamer instead of Oligo(dT)18 should be used for the reverse transcription of bacterial total RNA which does not have poly(A) tails. Gene-specific primers used for reverse transcription must be well designed.f. For RNA template with high GC content or secondary structures, increase the reverse transcription temperature to 45-50℃ to improve the reverse transcription efficiency... Read More | Inquire | Fumarate hydratase-IN-2 sodium salt (compound 3) is a cell-permeable and competitive fumarate hydratase inhibitor ( K i =4.5 µM) with nutrient-dependent cytotoxicity.Appearance:SolidIC50& Target:Ki: 4.5 µM (Fumarate hydratase)Biological Activity:Fumarate hydratase-IN-2 sodium salt (Fumarate hydratase-IN-2 sodium salt (compound 3) is a cell-permeable and competitive fumarate hydratase inhibitor ( K i =4.5 µM) with nutrient-dependent cytotoxicity.Appearance:SolidIC50& Target:Ki: 4.5 µM (Fumarate hydratase)Biological Activity:Fumarate hydratase-IN-2 sodium salt (compound 3) is a cell-permeable and competitive fumarate hydratase inhibitor ( K i =4.5 µM) with nutrient-dependent cytotoxicity... Read More | Product introduction：Used to isolate lymphocytes from human organsMatters needing attention：1. samples, reagents and experimental environment in the whole process shall be carried out at 20 ± 2 ℃. In order to obtain the best experimental results, it is best to carry out the Product introduction：Used to isolate lymphocytes from human organsMatters needing attention：1. samples, reagents and experimental environment in the whole process shall be carried out at 20 ± 2 ℃. In order to obtain the best experimental results, it is best to carry out the experiment within 2 h of sampling. The longer the sample is stored, the worse the cell separation effect is. The separation effect is even worse after the sample is placed for more than 6 h, or even cannot achieve the purpose of separation. 2. in this experiment, it is better not to use plastic products with high polymerization materials (such as polystyrene), but use non-static, low static ionization heart tubes and glass products without alkali treatment, because the electrostatic effect will lead to cell adhesion, and the surface of alkali treated glass will become rough, which will affect the effect of cell separation. 3. aspirating too many lymphocyte layers and separation liquid layers will cause the granulocytes at the junction of separation liquid to be aspirated, thus increasing the number of mixed granulocytes. 4. when the amount of separating solution is greater than that of tissue single cell suspension sample, the separation effect is better.Scope of application：Lymphocyte isolation... Read More | This reagent kit is based on TRIzon's improved columnar total RNA extraction kit. This product can be extracted from animal groupsExtract total RNA from samples such as textiles, plant materials, various microorganisms, and cultured cells. Firstly, the cracking solution is fully cracked This reagent kit is based on TRIzon's improved columnar total RNA extraction kit. This product can be extracted from animal groupsExtract total RNA from samples such as textiles, plant materials, various microorganisms, and cultured cells. Firstly, the cracking solution is fully cracked andHomogenized samples, in their unique high salt state, RNA specifically binds to silicon matrix membranes, greatly reducingEffectively removing organic solvent contamination while removing protein contamination, resulting in higher purity and quality of RNA. bookThe product can quickly extract total RNA from various cells or tissues, and can process 30-50 mg of tissue or 5 × 10 ⁶ cells each time,Can handle multiple different samples simultaneously. If it is an RNA experiment that is very sensitive to trace amounts of DNA, the residual DNA can be utilizedUsing DNase without RNase for digestion and removal on the column, the extracted RNA can be directly applied to RT-PCR Experiments such as Northern Blot, Dot Blot, and in vitro translation. U665516 Component 50 T Storage U665516A DNase I 1000 U -20℃. Avoid freeze/thaw cycle. U665516B 10×Reaction Buffer 1000 µL -20℃. Avoid freeze/thaw cycle. U665516C TRIzon Reagent 60 mL 2-8℃. Protect from light. U665516D TRIzon PaI™ 10 mL 2-8℃. Protect from light. U665516E Buffer RW1 40 mL RT U665516F Buffer RW2 (concentrate) 11 mL RT U665516G RNase-Free Water 10 mL RT U665516H Spin Columns RM with Collection Tubes 50 sets RT U665516I RNase-Free Centrifuge Tubes (1.5 mL) 50 EA RTPreparation and important precautions before the experiment:1.To prevent RNase pollution, attention should be paid to the following aspects:1) RNase's plastic products and gun heads to avoid cross contamination.2) Prepare the solution using water without RNase.3) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.2. The sample should avoid repeated freezing and thawing, otherwise it will affect the yield and quality of RNA extraction.3. If TRIzon Reagent is found to have precipitates before use, it can be dissolved in a water bath at 56 ℃ for a few minutes.Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.5. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.Usage：1. Sample processing1a. Organization: 30-50 mg of tissue is thoroughly ground in liquid nitrogen and 1 mL of TRIzon Reagent is added, or 1 mL of TRIzon Reagent is added to the tissue sample and homogenized. Attention: The sample volume should not exceed 10% of the volume of TRIzon Reagent.2a. Single layer cell culture: Remove the culture medium and add an appropriate amount every 10 cm ² Add 1 mL of TRIzon Reagent.3a. Cell suspension: Collect cells by centrifugation. Add 1 mL of TRIzon Reagent to every 5 × 10 µ m cell.2. After adding TRIzon Reagent, repeatedly blow a few times to fully crack the sample. Leave at room temperature for 5 minutes to completely separate the protein nucleic acid complex.3. Add 200 to every 1 mL of TRIzon Reagent µ LTRIzon PaI ™， Cover the tube tightly, vigorously shake for 15 seconds, and let it sit at room temperature for 2 minutes.4. Centrifuge at 4 ℃ 12000 rpm (~13400 × g) for 10 minutes. At this time, the sample is divided into three layers: the red organic phase, the middle layer, and the upper colorless aqueous phase. RNA is mainly in the upper aqueous phase. Move the upper aqueous phase to a new RNase Free centrifuge tube (provided).5. Add an equal volume of 70% ethanol (prepared without RNase water) to the obtained aqueous solution, invert and mix well.6. Add all the solutions obtained in the previous step to the spin columns RM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred in multiple batches. Centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.8. Preparation of DNase I mixture: Take 52 µ LRNase Free Water, add 8 to it µ L 10 x Reaction Buffer and 20 µ L DNase I (1 U/ µ L) Mix well and prepare to a final volume of 80 µ The reaction solution of L.9. Directly add 80 µ L DNase I mixture to the adsorption column and incubate at 20-30 ℃ for 15 minutes.10. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and place the adsorption column back into the recovery manifold.11. Add 500 to the adsorption column µ L Buffer RW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.12. Repeat step 11.Centrifuge at 12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes and thoroughly air dry. Attention: The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (enzyme digestion,. )PCR, etc.14. Place the adsorption column 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 RNA 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 14 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 and repeat step 14... Read More |