Purification of DNA from living cells

Transcription

1 Purification of DNA from living cells Total cell DNA & Plasmid DNA Grow and harvest bacterial culture Prepare cell extract Purify DNA from a cell extract Concentrate DNA samples Measure DNA concentration

2 Grow and harvest a bacterial culture Liquid medium with the essential nutrients Undefiend medium : LB (Luria- Bertani) precise identity and quantity are not known Prepare a source of DNA LB Maximum density of about 2-3 x 10 9 cells/ml (LB, 37 o C, 250 rpm) One optical density at 600 nm corresponds to about 0.8 x 10 9 cells/ml.

3 Preparation of a cell extract The bacterial cell is enclosed in a cytoplasmic membrane and surrounded by a rigid cell wall. In the case of E.coli, the cell wall is enveloped by a second, outer membrane. How do we disrupt these barriers to release the cell components? Physical methods - mechanical forces. Chemical methods - chemical agents.

4 Chemical methods Chemical agent Lysozyme: digests the polymeric compounds to weaken the cell wall. EDTA: removes Mg ++ that are essential for preserving the overall structure of the cell envelop and also inhibits cellular enzymes that could degrade DNA. SDS: is a detergent that can help the process of lysis by removing lipid molecules and thereby cause disruption of the cell membrane. Final step removal of insoluble cell debris by centrifugation.

5 Purification of DNA from a cell extract Organic extraction and enzyme digestion Deproteinization Phenol/chloroform extraction the organic solvent precipitate proteins and leave the nucleic acids in solution. Protease treat the cell extract with Proteinase K before Phenol/chloroform extraction RNA Some mrna can be removed by phenol treatment The majority can be removed by ribonuclease

6 A common way of concentration is ethanol precipitation. In the presence of salt (monovalent cations such as Na + ), and at a temperature of -20 o C or less, absolute ethanol will efficiently precipitate polymeric nucleic acids. After precipitation, dissolve mini-prep DNA in an appropriate volume of TE (Tris-EDTA) solution and store at - 20 o C. Use ultraviolet absorbance spectrophotometry to accurately measure DNA concentration. The amount of UV radiation absorbed by a solution of DNA is directly proportional to the amount of DNA in the sample. Usually absorbance is measured at 260 nm, at which wavelength an absorbance (A 260 ) of 1.0 corresponds to 50 g of double-stranded DNA per ml.

7 Separation of plasmid DNA from chromosomal DNA Plasmid DNA can be separated from bacterial DNA based on physical differences size plasmid DNA is only 8% or less of the E.coli chromosome conformation overall spatial configuration of the molecules

8 Size fractionation lyses cells in a very carefully controlled conditions to obtain spheroplasts, wall-less cells EDTA, lysozyme and sucrose are use to partially degrade cell walls induce cell lysis by adding a nonionic detergent such as Triton X- 100 (ionic detergents, such as SDS, cause chromosomal breakage). This method causes very little breakage of the bacterial DNA, so centrifugation will now leave a cleared lysate, consisting almost entirely of plasmid DNA.

9 Conformation separation - Alkaline denaturation 1. Based on the fact that there is a narrow ph range at which non-supercoiled DNA is denatured, whereas supercoiled plasmids are not. 2. Add sodium hydroxide to a cell extract or clear lysate will adjust the ph to , then the hydrogen bonding in non-supercoiled DNA molecules will be broken, causing the double helix to unwind and the two polynucletide chains to separate. 3. Add acid to bring the ph back to neutral, allowing the bacterial DNA to renature. The large, disrupted chromosomal strands cannot rehybridize perfectly but instead collapse into a partially hybridized tangle. 4. Meanwhile, the potassium acetate precipitates the SDS from the cell suspension, along with the associated proteins and lipids. Chromosomal DNA will be trapped in this SDS/protein/lipid precipitate. 5. Centrifugation will pellet this insoluble network and leave plasmid DNA in the supernatant.