| Description | Product contentG665760Component5 mLStorageG665760A2×GoldStar Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.G665760B50×Low ROX200 µL-20℃. Avoid freeze/thaw cycle.G665760CddH₂O5×1 mL-20℃. Avoid freeze/thaw cycle. Product Introduction GoldStar Probe Product contentG665760Component5 mLStorageG665760A2×GoldStar Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.G665760B50×Low ROX200 µL-20℃. Avoid freeze/thaw cycle.G665760CddH₂O5×1 mL-20℃. Avoid freeze/thaw cycle. Product Introduction GoldStar Probe Mixture is a premixed system for real-time fluorescence quantitative PCR by probe method (TaqMan, Molecular Beacon, etc.), with a concentration of 2×, containing GoldStar Taq DNA Polymerase, PCR Buffer, dNTPs and Mg2+, which is easy and convenient to operate. It is mainly used for genomic DNA target sequence and RNA reverse transcription cDNA target sequence detection, such as gene expression analysis, copy number analysis, SNP genotype analysis, etc. It is suitable for fluorescence quantification by different types of probe methods. GoldStar Taq DNA Polymerase is a chemically modified, new and highly efficient hot-start enzyme, which has no polymerase activity at room temperature and effectively avoids non-specific amplification caused by non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme must be incubated at 95℃ for 10 minutes. The unique combination of PCR buffer system and hot-start enzyme significantly improves the amplification efficiency of PCR with stronger fluorescent signal and higher sensitivity to detect single-copy templates. A wider linear range and more accurate quantification of target genes can be obtained with this product. It is suitable for all fluorescent quantitative PCR instruments that do not require ROX as a calibration dye.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and others. 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 (G665832) :Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.Instruments requiring Low ROX calibration (G665832) :ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA7 system. Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and more.Instruments requiring High ROX calibration (G665832) :ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1. Before use, please mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.2. Avoid repeated freezing and thawing of this product, repeated freezing and thawing may degrade the product performance. This product can be stored for long term at -20℃, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.UsageThe following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.1.PCR reaction systemreagents50µl reaction systemfinal concentration2×GoldStar Probe Mixture25µl1×Forward Primer, 10µM1µl0.2µM¹⁾Reverse Primer, 10µM1µl0.2µM¹⁾Probe, 10µM1µl0.2µM²⁾Template DNA2µl³⁾ 50xLow ROX or High ROX(optional)⁴⁾1µl1×ddH₂OUp to 50µlNote: 1) Usually, the primer concentration of 0.2µM can get better results, and 0.1-1.0µM can be used as a reference for setting the range. 2) The concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the instruction manual of the instrument or the specific requirements of the use of each fluorescent probe for the adjustment of the concentration during the actual use.(3) Usually the amount of DNA template is 10-100ng genomic DNA or 1-10ng cDNA as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be subjected to gradient dilution to determine the optimal amount of template to be used.(4) 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.2.PCR reaction programCaution! The pre-denaturation reaction of this product must be completed at 95°C for 10 minutes!Two-step PCR::Note: 1) The hot start enzyme used in this product must be activated under the condition of pre-denaturation 95℃, 10min. 2) It is recommended to use two-step PCR reaction program, if you can't get good experimental results due to the use of primers with lower Tm value, etc., you can try to carry out a three-step PCR amplification, and the annealing temperature should be set in the range of 56℃-64℃ as a reference... 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 | Inquire | Inquire | Reverse transcriptases are enzymes encoded in retroviruses viral genome. The enzyme is responsible for transcription of the viral RNA to produce a dsDNA that can be inserted into the host genome.Reverse transcriptases are multifunctional enzymes. These enzymes exhibit an RNA and DNA directed Reverse transcriptases are enzymes encoded in retroviruses viral genome. The enzyme is responsible for transcription of the viral RNA to produce a dsDNA that can be inserted into the host genome.Reverse transcriptases are multifunctional enzymes. These enzymes exhibit an RNA and DNA directed polymerase activity. In addition reverse transcriptases catalyze the degradation of RNA in an RNA-DNA hybrid. The exonucleolytic activity proceeds in a 5' ---> 3' direction. The RNA or DNA directed activity requires a template (RNA or DNA) and a primer. The following is a schematic illustration of the reaction:Unit definition: One unit incorporates 1 nanomole of tritiated dTMP into acid insoluble productsusing poly(A)•oligo(dT) 12-18 as the template-primer in 20 minutes at 37° C.ApplicationsHIV reverse transcriptase is used for research on the AIDS primer. However it can be substituted for AMV reverse transcriptase, which is mainly used to transcribe mRNA into double stranded cDNA, that can be inserted into prokaryotic vectors. The enzyme can also be used with either single stranded DNA or RNA templates to make probes for use in hybridization experiments. It can be used for labeling the termini of DNA fragments with protruding 5' termini. The enzyme can also be used to sequence DNAs by the dideoxy chain termination method of Sanger when the Klenow fragment of E. coli DNA polymerase I, or the T7 DNA polymerase yield unsatisfactory results.Reagents0.05 M Tris, pH 8.3, containing 0.008 M MgCl21 mg/ml polyadenylic acid in water (poly A)DNA primer:Oligo d(T)12-181 µ mole dTTP/mL stock solution[methyl-3H]-Thymidine 5'-triphosphate (3H-dTTP)dTTP-3H-dTTP working mix: Add 1-2 µL 3H-dTTP per mL of 100 nmol/mL dTTP in order to obtain 1 to 1.5 x 105 cpm/mL1% bovine serum albumin10% perchloric acid1% perchloric acidBuffer substrate reaction mixture: Prepare fresh, immediately before use:For each 1mL of reaction mixture required mix:0.7 mL Tris/HCl, pH 8.3, 0.008M MgCl20.3 mL 1 mg/mL poly(A) RNA template0.005 mL 0.02 mg/mL oligo d(T)12-18 DNA primer0.02mL 1% BSAEnzymedilute as needed wtih 0.05M Tris/HCl, pH 8.3, 0.008M MgCl2 containing 0.1 mg/mL (1%) BSAProcedurePipette into each tube as follows:Buffer substrate mix：0.1 mLdTTP-3H3-dTTP：0.1 mLEnzyme：5-10 µLIncubate 20 minutes at 37° C. Stop reaction by adding 1 ml 10% cold perchloric acid. Filter through 0.2µ manifold filters used with Millipore vacuum manifold. Wash four times using 2mL 1% cold perchloric acid/wash. Transfer filter to scintillation vials. Add 2mL Cellosolve (or 2-methoxyethanol) to dissolve filter. Filters become opaque upon addition of Cellosolve. Make sure filters are dissolved before proceeding. Add 10mL scintillation cocktail and count.Calculation... Read More |