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Preparation method of DNA pretreatment buffer liquid, thallus DNA and nucleic acid DNA

A technology of buffer solution and bacteria, applied in sugar derivatives, organic chemistry, etc., can solve the problems of high cost, difficult DNA extraction, and large sample loss

Inactive Publication Date: 2008-04-23
HARBIN INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Aiming at the difficulty of extracting DNA from environmental microbial samples, the yield is low, or even impossible to extract, the sample loss is large, the cost is high, and the problems that cannot fully and truly reflect the types and quantities of microorganisms in environmental samples, the present invention provides a Environmental microbial samples (sewage, bioreactor sludge, etc.) are directly used in PCR reaction DNA pretreatment buffer and bacterial DNA, nucleic acid DNA preparation method, which has low cost, fast, can obtain high-quality DNA, obtain The advantages of DNA can truly reflect the number and types of microorganisms in environmental samples

Method used

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Experimental program
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Effect test

specific Embodiment approach 1

[0011] Specific embodiment one: the composition and drug concentration of the DNA pretreatment buffer of the present embodiment are: 10× stock solution (70gNaCl; 2gKCl; 12gNaCl 2 HPO 4 ;2gKH 2 PO 4 ) and SDS, the addition of the SDS is 1‰ of the stock solution quality, the working solution is a 10-fold diluted DNA pretreatment buffer, and the pH is 7.5.

specific Embodiment approach 2

[0012] Specific embodiment two: the rapid preparation of the present embodiment is directly used in the method for the bacterial cell DNA of PCR amplification to realize through the following steps (taking petroleum sewage as example, OD value (absorbance)≤0.1): a, the following steps in Completed under the ultra-clean workbench: the workbench is first sterilized by ultraviolet light for 20-40 minutes, and then sampling: take 1mL of the water sample of oilfield sewage retrieved by an anaerobic tube containing high-purity nitrogen and inject it into the sterilized 1.5mL In a centrifuge tube; b. Fully shake on the shaker for 5 minutes, centrifuge at 13000r / min for 1 minute, and gently discard 0.9mL of supernatant from the tube; c. Add DNA pretreatment buffer 0.9 to the remaining 0.1mL water sample mL, shake fully on a shaker for 5 minutes; d, centrifuge at 13,000 r / min for 2 minutes, discard 0.98 mL of the supernatant, shake fully for 2 minutes, and obtain cell DNA after preparat...

specific Embodiment approach 3

[0013] Specific implementation method three: for the sludge treatment in the bioreactor, the method of gradient centrifugation is mainly adopted, and the specific steps are: a, the following steps are completed under the ultra-clean workbench, and the ultraviolet ray sterilization workbench is 20-40min, sampling: will use Take 0.1mL of the water sample of the reactor sludge retrieved from the sterilized tube and inject it into a sterilized 1.5mL centrifuge tube, add 0.9mL DNA pretreatment buffer at the same time, and fully shake on the shaker for 5min; b, Centrifuge at 3600r / min for 1min, collect the supernatant from the tube and transfer it to another 1.5mL centrifuge tube; c. Add the same volume of DNA pretreatment buffer as the supernatant, and shake again on the shaker for 5 minutes; d. Repeat steps b and c; e, centrifuge at 13,000 r / min for 2 min, discard the supernatant, add 20 μL of DNA pretreatment buffer into the tube, shake fully on the shaker for 5 min, and obtain ba...

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Abstract

A buffer liquid for the pre-treating of DNA is prepared from storage liquid and SDS. A process for preparing the DNA of sewage thallus includes oscillating, centrifuge, removing supernatant, adding said buffer liquid, oscillating, centrifuge, removing supernatant, and oscillating. A process for preparing nucleic acid DNA includes preparing thallus DNA and PCR amplification. Its advantages are high speed and low cost.

Description

technical field [0001] The invention relates to a method for preparing DNA pretreatment buffer, bacterium DNA and nucleic acid DNA for direct use of environmental microorganism samples in PCR reaction. Background technique [0002] At present, most of the methods used to prepare DNA from sewage, sludge, etc. in environmental samples are artificial preparation of drugs, using lysozyme ("A large-scale extraction method of bacterial DNA" Microbiology Bulletin. 1991.18 (1): 47-50) , repeated freezing and thawing combined with CTAB ("Research on the extraction method of total DNA from activated sludge that can be used in the study of molecular ecology of microbial communities" Acta Ecologica Sinica 2002, 22(11): 2015-2019) or sodium dodecylsulfonate SDS method ( "Research on the Extraction Method of Total DNA from Activated Sludge" Biotechnology 2005, 15(4): 43-45.) to extract the DNA of microorganisms in the sample, or prepare it with a kit, the cost is high and the DNA extracti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07H21/04
Inventor 魏利马放
Owner HARBIN INST OF TECH