PicoGreen dsDNA Assay Kit (C09-1009-425)

Product descriptionPicoGreen fluorescent dye for dsDNA quantitative detection1. Application descriptionIn molecular biology experiments, the PicoGreen dsDNA quantification kit is a product for fluorescent detection and quantification of double-stranded DNA. This method is very sensitive. Commonly used in molecular biology technology: cDNA library construction; DNA fragment purification and application for subcloning, such as DNA quantification, product amplification, and further primer detection. Vaccines are a commonly used control method in modern disease prevention. Many vaccines today are cell culture vaccines, such as recombinant hepatitis B vaccine, rabies vaccine and most of the vaccines are produced using cell culture methods. Among them, the purification of vaccines is a key issue, and we need to remove host cell DNA and host proteins as much as possible. If the host cell's DNA and protein are injected into the human body together with the vaccine, it will have unpredictable consequences.The conventional DNA content detection method is to measure its absorbance at 260nm (A260). The main disadvantage of this method is that nucleotides, single-stranded nucleic acids and proteins have a great influence on the signal, and are also interfered by contaminants in the nucleic acid preparation process, and cannot distinguish between DNA and RNA, and this method is not sensitive (5μg) /mLdsDNA solution A260=0.1). The PicoGreen quantitative detection method is simple and convenient, and has been selected by many biological product manufacturers, and has become the standard for the detection of residual DNA in biological products. At present, this method has been included in the 2010 edition of "Chinese Pharmacopoeia"Principle:The fluorescence emitted after PicoGreen is combined with the double strand of DNA, without DNA, does not emit fluorescence; the fluorescence emitted is proportional to the concentration of DNA. It was proposed in the Chinese Pharmacopoeia in 2010 that the detection limit of PicoGreen method for quantifying DNA is about 0.3ng/ml, and the linearity is better when the DNA content is in the range of 1.25-80ng/mL (R2>0.99).Advantage:1) This method can measure double-stranded DNA in samples derived from any expression host.2) It is possible to directly quantify PCR products without purifying DNA from the reaction mixture.3) Far beyond the traditional UV A260 detection method and the sensitivity of Hoechst33258.4) Higher concentrations of salt, urea, ethanol, chloroform, detergent, protein or agarose have no effect on the determination.5) Measuring dsDNA in the presence of equimolar concentrations of ssDNA and RNA has little effect.2. Required equipment• Mini Fluorometer; Portable Fluorometer-Shanghai Hubei Scientific Instrument Co., Ltd. HG-9; 1cm quartz cuvette • PicoGreen dsDNA Quantitative Detection Kit, a 1mL unit volume of reagent concentrate is sufficient for 200 determinations in a volume of 2mL. 1×TE (10mM Tris 1mM EDTA) pH8.0; 250ug/mL calf thymus DNA3. Experimental protocolReagent preparationPicoGreen dsDNA quantification reagent is stored in anhydrous DMSO (dimethyl sulfoxide) in the form of a 1mL concentrated solution. On the day of the experiment, prepare the operating solution of 2XPicoGreen reagent and dilute the concentrated solution (10mM Tris-HCl, 1mM EDTA, pH7.5) with 1xTE at a ratio of 1:200. If you want to prepare enough operating solution to measure 20 samples, you can add 100μL PicoGreen dsDNA quantification reagent to 20mL1x TE. Since the reagent is easily adsorbed to the glass surface, it should be prepared in a plastic container. PicoGreen reagent is easily degraded when exposed to light, so the prepared solution should be wrapped in foil or stored in a dark place away from light.The solution is best used within a few hours of preparation to ensure the best results.Experimental method:1). Preparation of standard working solution: calf thymine DNA dry powder 1mg (Tris, Nacl and other concentrations have become standard systems), add 1mL of double distilled water to prepare 1mg/mL standard working solution ;2). Dye working solution configuration:6 Add 1mL TE to uLPicoGreen (Note: Dilute PicoGreen 200 times with 1×TE, and use it now, please avoid light).3). Standard working solution dilution:(1) Mother liquor dilution: Take 10ul (1mg/mL) standard working solution and add it to 990ul TE solution, dilute to 10ug/mL, take 10ul (10ug/mL) standard working solution and add it to 990ul TE solution, concentration Dilute to 100ng∕mL;(2) Multiple dilution: Take 800ul (100ng/mL) standard working solution and add it to 200ul TE solution, the concentration reaches 80ng/mL (pharmacopoeia stipulates: the DNA content of fluorescent staining method is linear in the range of 1.25-80 ng/mL Ok, the DNA detection limit of this method is 0.3 ng/ml). Take 500ul (80ng/mL) of standard working solution and add it to 500ul TE solution, and dilute to 40ng/mL; successively dilute by multiples and make 20ng/mL. Standard solution of ml, 10ng/ml, 5.0ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml;4). Preparation of standard curve: take 100ul of each gradient standard solution and dye working solution diluted by multiple ratios and mix them evenly, and place them at room temperature in the dark for 5 minutes. Use the FB-15 portable fluorometer to detect the fluorescence value of the sample: add the mixed solution into the micro cuvette, make sure not to introduce air bubbles into the sample, and gently flick the outside of the micro detection cuvette to disperse the air bubbles. Use 1×TE buffer as blank to measure the fluorescence values of the sample and the blank control; use the fluorescence intensity corresponding to the concentration of the standard solution (ng/ml) to make a linear regression to prepare a standard curve.5). Measure the fluorescence value of the remaining samples. The fluorometer will give a direct concentration reading, and the data can be used to generate a standard curve of DNA concentration.Product parameterEx(nm) 488        Em(nm) 520ComponentMainly including three components: dye, standard, and buffer. Related Document: https://ald-pub-files.oss-cn-shanghai.aliyuncs.com/aladdinsci/pdp/sds/1/P266245-SCI_ebd83fe3ff2a8596572f76f3f46edb22.pdf.