酚氯仿法提取DNA原理及方法

Chapter 1 DNA extraction

说明：本原理及方法是个人整理，用来给研究生教学用的，用本方法可以提取到理想的DNA。各实验室提供的细胞裂解液浓度各有不同酚氯仿法提取dna的步骤，只要经过实验证实的，都可以用来提取到理想的DNA.

1.Experimental Principles

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SDS, a detergent is added to the buffer to break open the cell membranes; it also helps remove proteins and lipids in the cell. Ethylenediaminetetraacetic acid (EDTA), a chelator to remove metal ions in solution to preven DNase from cutting up the DNA. RNase is also present in the buffer at this step, to break up the RNA present in the cells.

2)Remove protein

Proteinase K, it remains active at elevated temperatures, so the solution can be heated to about 55 °C to aid protein inactivation and removal by the detergent.

3)Extract DNA from buffer

Once the cells are broken open and the RNA, proteins, and lipids have been dissolved in the buffer, the DNA must be separated from these materials.

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4)DNA precipitation

The ethanol can precipitate DNA from water phase

2.Materials and Solutions

All reagents are precooled or kept at 4°C before use.

1)Proteinase K

2)Phenol saturated with TE (pH 8.0)

3)Chloroform

4)Isoamyl alcohol

5)RNase stock (30 mg/ml, Catalog No.R4642-10MG, Sigma )

6)10% SDS

7)0.5 mol/L EDTA, PH=8.0

8) 1 mol/L Tris-HCl, PH=8.0

9) 1 mol/L NaCl

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8.0), 0.5% SDS, 50 μg/ml RNase)