Description
In the experimental process of molecular biology, Fluo ™ Green ® The dsDNA quantification kit is a product used for fluorescence detection and quantification of double stranded DNAThe method is very sensitive. Commonly used in molecular biology techniques: construction of cDNA libraries; Purification and application of DNA fragments for subcloning, such as DNA quantification, product amplification, and further detection of primers. Vaccines are a commonly used control method in modern disease prevention. Nowadays, many vaccines are cell culture vaccines, such as recombinant hepatitis B vaccine, rabies vaccine and most other vaccines are produced by cell culture. 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 DNA and protein of the host cell are injected into the human body together with the vaccine, unpredictable consequences will occur.The conventional method for detecting DNA content 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 significant impact on the signal, and are also subject to interference from pollutants during the nucleic acid preparation process, making it difficult to distinguish between DNA and RNA. Additionally, this method is insensitive (5 µ g/mL dsDNA solution A260=0.1). Fluo ™ The Green quantitative detection method is simple and convenient, and has been selected by multiple biological product factories, becoming the standard for residual DNA detection in biological products.At present, this method has been included in the 2010 edition of the Chinese PharmacopoeiaPrinciple:Fluo ™ Green emits fluorescence only after binding to double stranded DNA, and does not emit fluorescence without DNA; The fluorescence emitted is directly proportional to the concentration of DNA. In the 2010 Chinese Pharmacopoeia, it was proposed that, Fluo ™ The detection limit of Green's quantitative DNA method is about 0.3ng/ml, and the linearity is good (R2>0.99) when the DNA content is in the range of 1.25-80ng/mLAdvantage:1) This method can determine double stranded DNA from any expressed host sample.2) It is possible to directly quantify PCR amplification products without purifying DNA from the reaction mixture.3) Far exceeding the sensitivity of traditional UV A260 detection methods and Hoechst33258.4) Higher concentrations of salt, urea, ethanol, chloroform, detergents, proteins, or agarose have no effect on the measurement.5) The effect of measuring dsDNA in the presence of equimolar concentrations of ssDNA and RNA is minimal.Required equipment• Micro fluorescence meter; Portable fluorescence analyzer - Shanghai Huguo Scientific Instrument Co., Ltd. HG-9 model; 1cm quartz colorimetric dish• Fluo ™ Green dsDNA quantitative detection kit, 1mL of concentrated reagent solution is sufficient for 200 measurements of 2mL volume.1×TE（10mM Tris 1mM EDTA）pH8.0； 250ug/mL calf thymus DNAExperimental planPreparation of reagentsFluo ™ The Green dsDNA quantification reagent is stored in anhydrous DMSO (dimethyl sulfoxide) in the form of 1mL concentrated solution. On the day of the experiment, prepare2X Fluo ™ The operating solution of Green's reagent was diluted with 1xTE at a ratio of 1:200 in concentrated solution (10mM Tris HCl,1mM EDTA, pH 7.5). If you want to prepare enough operating solution to determine 20 samples, you can add 100 µ L Fluo to 20mL1x TE ™ Green dsDNA quantification reagent. Due to the easy adsorption of reagents onto glass surfaces, they need to be prepared in plastic containers. Fluo ™ Green reagent is easily degraded by light, so the prepared solution should be wrapped in foil or stored in a dark place away from light.It is best to use the solution within a few hours of preparation to ensure optimal results.Experimental method:1). Preparation of standard working fluid:1mg of calf thymidine DNA dry powder (Tris, NaCl, and other concentrations have become standard systems) is added to 1mL of double distilled water to prepare a 1mg/mL standard working solution;2). Configuration of dye working fluid:6 uL Fluo ™ Add 1mL of TE to Green (note: use 1 × TE to mix Fluo) ™ Dilute Green 200 times, use and prepare immediately, avoid light.3). Dilution of standard working fluid:(1) Mother liquor dilution: Take 10ul (1mg ⁄ mL) of standard working solution and add it to 990ul TE solution to dilute the concentration to 10ug ⁄ mL. Take 10ul (10ug ⁄ mL) of standard working solution and add it to 990ul TE solution to dilute the concentration to 100ng ⁄ mL;(2) Dilute by multiple ratio: Take 800ul (100ng ⁄ mL) of standard working solution and add it to 200ul of TE solution to achieve a concentration of 80ng ⁄ mL (pharmacopoeia regulation: fluorescent)The light staining method shows good linearity in the range of 1.25-80 ng/mL for DNA content, with a detection limit of 0.3 ng/mL. Take 500ul (80ng ⁄ mL) of standard working solution and add it to 500ul TE solution, diluting the concentration to 40ng ⁄ mL; Dilute sequentially by multiple ratios to prepare 20ng/ml 10ng/ml 5.0ng/ml 2.5ng/ml1.25ng/ml and 0.625ng/ml standard solution;4). Preparation of standard curve: Take 100ul of each gradient standard solution and dye working solution diluted by multiple ratios, mix well, and leave them at room temperature in the dark for 5 minutes. Use FB-15 portable fluorescence analyzer to detect the fluorescence value of the sample: Add the mixed solution to the microcalorimeter, make sure not to introduce bubbles into the sample, and gently tap the outside of the microcalorimeter to disperse the bubbles. Measure the fluorescence values of the sample and blank control using 1 × TE buffer as a blank; Corresponding to the concentration of standard solution (ng/ml)Perform linear regression on fluorescence intensity and prepare a standard curve.5). Measure the fluorescence value of the remaining samples. The fluorescence meter will provide a direct concentration reading, which can be used to generate a standard curve of DNA concentration. Final concentration of DNA to be tested Fluorescence reading value (ng/ml) / 100 6210 50 3195 40 2507 20 1261 10 620.8 5 298 4 258.8 2 152 0.5 43.8 0 0.72