Method for dyeing DNA in cesium chloride density gradient mixed liquor through GelGreen dye

A technology of density gradient and mixed solution, which is applied in the field of molecular biology to achieve the effect of verification accuracy, improvement of detection sensitivity and high sensitivity

Active Publication Date: 2015-07-08
BEIJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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[0005] At present, there is no related research on the application of

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  • Method for dyeing DNA in cesium chloride density gradient mixed liquor through GelGreen dye
  • Method for dyeing DNA in cesium chloride density gradient mixed liquor through GelGreen dye

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Embodiment 1

[0040] Prepare 1mL mixture of 5μg environmental total DNA and TE buffer, add 1.3g CsCl solid, vortex and mix well, add 4.7mL 1g / mL CsCl solution, add 8.85μL 10000X GelGreen dye to TE buffer treatment. The buoyant density of the prepared environmental total DNA-cesium chloride mixture was measured with an AR200 hand-held refractometer, and adjusted with 1g / mL CsCl solution and TE buffer, so that the buoyant density of the mixture was 1.72g / mL. Take 5.1 mL of the mixed solution and put it into an ultra-high-speed centrifuge tube, and centrifuge for 44 hours at 20°C and a centrifugal speed of 45,000 rpm. After the ultra-high-speed centrifugation, the density gradient mixture was irradiated by the fluorescence transilluminator, and the density gradient mixture in the ultra-high-speed centrifuge tube was divided into 4 parts based on the fluorescent DNA of the target layer. Layers I, II, III, and IV, in which the target band is in layer II, are drawn out sequentially from top to bo...

Embodiment 2

[0045] Prepare 5 μg of environmental DNA and GB buffer solution into 1 mL mixture, add 1.3 g of CsCl solid, vortex and mix well, then add 4.7 mL of 1 g / mL CsCl solution, and then add 8.85 μL of 10000X GelGreen dye to GB buffer solution. The buoyant density of the prepared environmental total DNA-cesium chloride mixture was measured with an AR200 hand-held refractometer, and adjusted with 1g / mL CsCl solution and GB buffer, so that the buoyant density of the mixture was 1.72g / mL. Take 5.1 mL of the mixed solution and put it into an ultra-high-speed centrifuge tube, and centrifuge for 44 hours at 20°C and a centrifugal speed of 45,000 rpm. After the ultra-high-speed centrifugation, the density gradient mixture was irradiated by the fluorescence transilluminator, and the density gradient mixture in the ultra-high-speed centrifuge tube was divided into 4 parts based on the fluorescent DNA of the target layer. Layers I, II, III, and IV, in which the target band is in layer II, are d...

Embodiment 3

[0051] Repeat the operation process of embodiment 1.

[0052] Observe the repeatability experiment results of DNA staining with GelGreen dye in TE buffer. In the repeatability experiment, the staining effect of GelGreen dye on DNA is consistent, indicating that the staining effect of GelGreen dye on DNA in the cesium chloride density gradient mixture has good repeatability.

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Abstract

The invention relates to a method for dyeing DNA in cesium chloride density gradient mixed liquor through GelGreen dye, and belongs to the field of molecular biological technologies. The method includes the steps that step1, an environmental sample is collected, and the environmental total DNA is extracted; step2, environmental total DNA-cesium chloride mixed liquor is prepared, and the GelGreen dye is added into the environmental total DNA-cesium chloride mixed liquor; step3, ultrahigh-speed centrifugation is performed on the mixed liquor with the GelGreen dye to obtain the density gradient mixed liquor; step4, layering is performed on the density gradient mixed liquor under irradiation of a fluorescence transmission instrument with the excitation wavelength of 488 nm, and the density gradient mixed liquor in corresponding layers is extracted through an injector; step5, bacterium 16S rRNA gene real-time fluorescence quantified PCR analysis is performed on the DNA in the density gradient mixed liquor in all the layers. By dyeing the DNA in the cesium chloride density gradient mixed liquor based on the GelGreen dye, the detection sensitivity of the environmental DNA in the cesium chloride density gradient mixed liquor obtained after ultrahigh-speed density gradient centrifugation is greatly improved.

Description

technical field [0001] The invention relates to a method for staining DNA with GelGreen dye in cesium chloride density gradient mixed solution, belonging to the technical field of molecular biology. Background technique [0002] Stable isotope probing (SIP) is a method of cultivating environmental microorganisms by using stable isotopes as specific substrates, so that microorganisms can bind stable isotopes to nucleic acids (DNA and RNA) and phospholipid fatty acids (PLFA) and other specific biomarkers, and then the biotechnology that couples the characteristics of microorganisms with their special metabolic functions. Compared with DNA-SIP, the disadvantage of PLFA-SIP is that most uncultured microorganisms lack PLFA molecular patterns, and it is difficult to link PLFA obtained from marker culture with species information. For RNA-SIP, due to the low amount of mRNA in environmental samples, it is difficult to recover sufficient mRNA for downstream analysis after ultrahigh-...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 高景峰潘凯玲樊晓燕司春英李洪禹李婷孙丽欣
Owner BEIJING UNIV OF TECH
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