| Description | Product contentU665751Component100 TStorageU665751A2×UltraSYBR One Step Buffer1.4 mL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751BUltraSYBR One Step EnzymeMix50 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751C50×High ROX50 µL-20℃. AvoidProduct contentU665751Component100 TStorageU665751A2×UltraSYBR One Step Buffer1.4 mL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751BUltraSYBR One Step EnzymeMix50 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751C50×High ROX50 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751DRNase-Free Water1.5 mL-20℃. Avoid freeze/ Thaw cycle. Product Introduction This product is a specialized kit for one-step Real-Time RT-qPCR. The SYBR Green I fluorescent dye contained can bind to all double-stranded DNA, allowing this product to be used for the detection of many different target sequences without the need to synthesize specific labeling probes. Real Time RT-qPCR reaction using this product, reverse transcription and quantitative PCR are carried out in the same reaction system, there is no need to add reagents during the reaction, no need to open the cap of the tube, avoiding contamination while improving the efficiency of the experiment. The new high-efficiency reverse transcriptase RNase H is activity-deficient, which reduces the degradation of RNA in the reverse transcription reaction. The enzyme has high reverse transcription efficiency and can perform a good reverse transcription reaction on a small amount of RNA template. It has high affinity to RNA and can read through RNA templates with high GC content and complex secondary structure. New efficient hot start enzyme, the enzyme activity is closed at room temperature, thus effectively avoiding non-specific amplification caused by non-specific binding of primers and templates or primer dimerization at room temperature, which greatly improves the accuracy of fluorescence quantitative PCR reaction. The included buffer system maximizes the efficacy of both enzymes at the same time and improves efficiency. This product has high sensitivity, high specificity, wide linear range, and more accurate quantification of target genes.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used with Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument. Instruments that do not require ROX calibration (U665567) Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others. Instruments that require High ROX calibration (U665751) ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1. Before using the reagents in this kit, please mix them gently by turning them up and down to avoid foaming as much as possible, and use them after brief centrifugation.2. This product uses RNA as the template for one-step RT-PCR experiment, RNase contamination should be avoided during operation, it is recommended to operate RNA in a special area, use special instruments and consumables, the operator with a mask and disposable gloves and often change the gloves, the experiment-related consumables should be processed with 0.1% DEPC (diethyl ether of pyrocarbonate) aqueous solution at 37℃ for 12 hours and autoclaved for 30 minutes before use. Sterilize for 30 minutes before use.3. UltraSYBR One Step RT-qPCR Buffer contains SYBR Green I fluorescent dye. Avoid bright light when storing this product or preparing PCR reaction solutions.4. Repeated freezing and thawing of each reagent in this kit should be avoided; repeated freezing and thawing may degrade the product performance. This product can be stored for a long time at -20℃, protected from light. If frequent use is required in the short term, it can be stored at 2-8℃.5. This kit must use specific primers, the choice of primers can be selected according to specific experiments, the good or bad primer design directly affects the results of RT-PCR reaction, the design of primers need to consider the GC content, primer length, primer position, the secondary structure of the PCR product and other factors, it is recommended to use a professional primer design software for design.6. This product cannot be used for fluorescent quantitative PCR by the probe method.Usage1. Dissolve RNA template, primers, 2× UltraSYBR One Step Buffer, UltraSYBR One Step EnzymeMix and RNase-Free Water and set aside on ice.2. PCR reaction system:Reagents25 µl Reaction systemFinal concentration2×UltraSYBR One Step Buffer12.5 µl1×Forward Primer，10 µM0.5 µl0.2 µM¹⁾Reverse Primer，10 µM0.5 µl0.2 µM¹⁾UltraSYBR One Step EnzymeMix0.5 µl RNA TemplateX µl10 pg – 100 ng50×Low ROX or High ROX（optional）2）0.5 µl1×RNase-Free Waterup to 25 µlNote: 1) Usually, the primer concentration of 0.2µM can get better results, and the final concentration of 0.1-0.5µM can be used as a reference for setting the range. If the amplification efficiency is not high, the concentration of primer can be increased; when non-specific reaction occurs, the concentration of primer can be decreased, thus optimizing the reaction system.(2) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.3. Vortex and shake to mix, centrifuge briefly, and collect the solution at the bottom of the tube.4. RT-qPCR reaction conditions (fluorescence quantitative PCR is a two-step method), this program is based on the ABI 7500 fluorescence quantitative PCR instrument as an exampleNote: 1) It is recommended to use two-step PCR reaction program, if you improve the reaction specificity, you can increase the annealing temperature to 60-64 ℃ as a reference for the setting range; if you do not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out three-step PCR amplification.(2) For melting curve analysis, please set up the program recommended by the fluorescence quantitative PCR instrument used, and this program is set up with the ABI 7500 fluorescence quantitative PCR instrument as a reference.RT-qPCR reaction conditions (fluorescence quantitative PCR was a three-step method):Note: 1) For three-step PCR amplification, please use the range of 56℃-64℃ as the setting reference for the annealing temperature.(2) For melting curve analysis, please set up the program recommended by the fluorescence quantitative PCR instrument you are using, this program is ABI750 fluorescent quantitative PCR instrument as a reference setting... 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 | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Product content Q665687Component100 TStorageQ665687AQuick T4 DNA Ligase (15 U/µL)100 µL-20℃. Avoid freeze/thaw cycle.Q665687B2×Quick Ligation Reaction Buffer5×200 µL-20℃. Avoid freeze/thaw cycle. Product IntroductionThe Quick Ligation Reaction Kit allows ligationProduct content Q665687Component100 TStorageQ665687AQuick T4 DNA Ligase (15 U/µL)100 µL-20℃. Avoid freeze/thaw cycle.Q665687B2×Quick Ligation Reaction Buffer5×200 µL-20℃. Avoid freeze/thaw cycle. Product IntroductionThe Quick Ligation Reaction Kit allows ligation of DNA sticky or flush ends in 5 minutes at room temperature (25°C). The kit contains Quick T4 DNA Ligase and 2×Quick Ligation Reaction Buffer optimized for fast and efficient DNA ligation.The ligation efficiency of Quick Ligation is equivalent to 1 hour of conventional ligation with T4 DNA Ligase. The Quick Ligation products can be used directly in routine bacterial transformation experiments.matters needing attention1. This kit enables most of the linkage reactions to reach the reaction endpoint within 5 minutes or less at 25°C. Increasing the reaction time will not enhance the reaction efficiency. If you use the rapid connection reaction after 1 hour, the conversion efficiency will be significantly reduced; if the rapid connection reaction at 25 ℃ overnight, the conversion efficiency will drop to 75%.2. 2×Quick Ligation Reaction Buffer contains ATP, which should be thawed on ice and mixed thoroughly before use. It is recommended to freeze the buffer in small tubes for the first time, so as to avoid repeated freezing and thawing, which will affect the efficiency of DNA ligation.3. Since T4 DNA Ligase contains glycerol, which is sticky and easy to hang on the wall, it is recommended to collect the liquid to the bottom of the tube by centrifugation for a short period of time before use, and the tip of the lance should not go too deep into the liquid surface when taking samples to avoid sticking to the tip of the lance and causing losses.4. If the quick ligation product is used for electrotransformation, the PEG in the quick ligation reaction system will affect the efficiency of electrotransformation, and it is recommended to use a centrifugal column to purify the ligation product from DNA before electrotransformation.Usage1. The reaction solution was prepared according to the following system:*The amount of Insert DNA used: the molar ratio of Vector DNA and Insert DNA is generally 1:3-1:8, and the appropriate molar ratio of Vector DNA and Insert DNA can be selected according to the experimental situation.Calculation of DNA molar number: DNA molar number (nmol)=DNA mass (ng)/( 660daltons x number of inserted DNA bases bp).2. mix gently and centrifuge briefly. react at 25°C for 5 minutes.Note: The reaction time should not exceed 15 minutes, otherwise the connection efficiency will be reduced.3. Do not perform heat inactivation reactions. Centrifuge instantly and collect the solution from the wall to the bottom of the tube.Note: Heat inactivation significantly reduces transformation efficiency due to the presence of PEG in the buffer.4. After the reaction, store the DNA ligation product at 0-4℃, and then carry out transformation experiments; you can also store the DNA ligation product at -20℃.Note: When transforming by chemical method, do not add more than 10% of the volume of the receptor cell for the ligation product.5. Heat shock the ligation product to transform 50 µl of receptor cells or take 1-2 µl of ligation product to electroshock transform 50 µl of receptor cells.Note: 1) When transforming by chemical method, do not add more than 10% of the volume of the receptor cell for the ligation product.(2) If the quick ligation product is used for electrotransformation, it is recommended to use a centrifugal column to purify the ligation product from DNA before electrotransformation because the PEG in the quick ligation reaction system will affect the efficiency of electrotransformation... Read More | The Succinic Acid (Succinate) assay kit is suitable for the specific assay of succinic acid in wine, cheese, eggs, sauce and other food products. Succinic acid (or succinate) is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the The Succinic Acid (Succinate) assay kit is suitable for the specific assay of succinic acid in wine, cheese, eggs, sauce and other food products. Succinic acid (or succinate) is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the Citric Acid Cycle. Succinic acid concentrations are monitored in the manufacture of numerous foodstuffs and beverages, including wine, soy sauce, soy bean flour, fruit juice and dairy products (e.g. cheese).Product Description： Succinic acid is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the Citric Acid Cycle. Succinic acid concentrations are monitored in the manufacture of numerous foodstuffs and beverages, including wine, soy sauce, soy bean flour, fruit juice and dairy products (e.g. cheese). The ripening process of apples can be followed by monitoring the falling levels of succinic acid. The occurrence of > 5 mg/kg of this acid in egg and egg products is indicative of microbial contamination. Apart from use as a flavouring agent in the food and beverage industries, succinic acid finds many other non-food applications, such as in the production of dyes, drugs, perfumes, lacquers, photographic chemicals and coolants. Preparation Instructions：Suitable for succinate determination in food, beverage, agricultural products, and other biological samples.Note for Content：The number of manual tests per kit can be doubled if all volumes are halved. This can be readily accommodated using the MegaQuantTM Wave Spectrophotometer (D-MQWAVE).Browse all of our organic acid assay kits.Principle：The Succinate Assay Kit provides a simple, one step assay for measuring succinate. In this assay succinate is converted to pyruvate which reacts with specific reagents and dye to form a colored product. The color intensity at 570 nm or fluorescencAdvantages：Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.Very competitive price (cost per test)All reagents stable for > 2 years as suppliedVery rapid reaction (even at room temperature)Mega-Calc™ software tool is available from our website for hassle-free raw data processingStandard includedSuitable for manual, microplate and auto-analyser formats... Read More |