Lu Quang c USTHBST2 016

A/Introduction
Plasmid is a small autonomously replicating genetic element, that can eplicate independently of the chromosomal DNA. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms and they are generally circular DNA molecules. Plasmids were historically used to genetically engineer the embryonic stem cells of rats in order to create rat genetic disease models To purify DNA plasmid from mixture of proteins, lipids, nucleicacids extraction is an esay and quick way Plasmid DNA is extracted from bacterial cells based on the alkaline lysis procedure which developed by Birnboim and Doly (Nucleic Acids Research, 1979, 7: 1513-1523). The procedure takes advantage of the fact that plasmids are relatively small supercoiled DNA molecules and bacterial chromosomal DNA is much larger and less supercoiled. This difference in topology allows for selective precipitation of the chromosomal DNA and cellular proteins from plasmid and RNA molecules. The cells are lysed under alkaline conditions (NaOH, SDS), which denatures both DNAs and proteins. When the solution is neutralized by the addition of Potassium acetate, chromosomal DNA and proteins precipitate because it is impossible for them to renature correctly (they are so large). Plasmids renature corectly and stay in solution. Plasmids can be selected by high speed centrifugation. RNA can be removed by enzyme RNase.

B/Materials

Solution I (Sol I): 100 ml Composition Volume Final concentration 50mM 25mM 10mM

Glucose 0,9 g Tris-HCl 1M, pH8 2,5 ml EDTA 0,5M, pH8 2 ml Add bidistilled water (ddH2O) to 100 ml Solution II (Sol II): 12 ml Composition SDS 10% NaOH 3M Sterilized ddH2O Volume 1,2 ml 0,8 ml 10 ml

Final concentration 1% SDS 0,2N NaOH

Solution III (Sol III): 100 ml of 3M Potassium Acetate Composition Volume CH3COOK 29,4 g CH3COOH 11,5 ml Add bidistilled water (ddH2O) to 100 ml Caution: Solution I and Solution III must be autoclaved and stored at 4oC. Solution II should be made fresh, non-autoclaved and stored at room

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temperature. Solution 24:1 (chloroform: isoamyalcohol) : 50 ml Composition Chloroform Isoamyalcohol Volume 48 ml 2 ml

Tris-acetate-EDTA (TAE) buffer ( for electrophoresis)

- Stock solution 50X TAE (for 500ml):

121g Trisbase in 250 ml ddH2O, stir to dissolve Add 28.6ml acetic acid Add 50ml 0.5M EDTA pH 8.0 Add ddH2O to 500ml - Working solution 1X TAE: The working solution of 1X TAE buffer is made by simply diluting the stock solution by 50X in deionized water. Other reagents:

Ethanol 100%, Ethanol 70% Agarose, Ethidium bromide

C/ Method and Observation

Day 1 (repaired) Inoculate a culture tube containing 2ml LB medium with single bacterial colony. Incubate at 37oC with shaking (200 rpm), overnight. Day 2 1.Transfer 1.5ml bacterial culture growth into a microcentrifuge tube (1.5ml Eppendorf tube). 2. Pellet bacterial cells by centrifuge at 6000 rpm for 8 min. (Fig 1) => after centrifuging the mixture is seperated into 2 parts: clear liquid (supernatant) and sediment (cell) Discard the supernatant. => take the cell of bacteria 3. Resuspend the cell pellet in 100 l Sol I by vigorous vortexing

Fig 1

(Fig 2) => Sol I contains: glucose trigger the rupture the cell wall, Tris used to maintain the constant pH EDTA protects the DNA from degration => aftering vortexing the liquid is mixtured completely and the cell wall is broken Fig 2

4. Place the resuspended cells on ice, and add 200 l of Sol II. Mix by gentle inversion several times => Alkaline pH from Sol II denatures chromosomal DNA but not are covalently closed circular plasmid DNA => form homogeneous mixtrue => the cell is completely lysed 5. Add 150 l of Sol III. Mix by gentle inversion. Incubate on ice for 5 min. => Sol III: CH3COOK used to precipitate choromosomes and proteins CH3COOH neutralizes the alkaline pH => Precipitation appears at the bottom of the tube including chromosomes, proteins 6. Centrifuge at 12000 rpm for 10 min. Carefully remove supernatant to a new tube, being careful not to transfer any of white debris in the pellet. => Chromosomes and proteins are precipitated and formed white debris => Plasmids return to the solution in the supernatant 7. Add 1l of RNase (10mg/ml) to the transferred supernatant. Incubate at 37oC for 1 hr. => to remove RNA in the solution 8. Extract the supernatant with an equal volume of Chloforform: Isoamyl (24:1), mix well and separate the upper aqueous phase by centrifuge at 12000 rpm at 4oC for 15 min.

=> add Chloforform: Isoamyl (24:1) to dissolve protein of cell membrane => after centrifuging the solution is seperated by a membrane in the middle The upper aqueous is the solution containing plasmid DNA 9. Pellet plasmid DNA with 100% Ethanol (add 2 volume of 100% Ethanol to 1 volume of the aqueous sample containing pladmis DNA), mix by inversion several times. Incubate at -20oC, for 1 hr. => enthanol is used to absord water and remove salts in solution and purify the plasmid DNA 10. Centrifuge at 12000 rpm, 4oC, for 15 min. Discard the supernatant => get the plasmid DNA 11. Wash the DNA pellet with 1ml of 70% Ethanol. Centrifuge at 12000 rpm, 4oC, for 15 min. Discard the supernatant (repeat the washing step once more time). 12. Dry the DNA pellet in a vacuum centrifuge (Speed-Vac) for 5 min. 13. Resuspend the DNA pellet completely in 20-30 l of sterile distilled water 14. Check the plasmid DNA by agarose gel electrophoresis (0.8% agarose gel)

Preparing and runing agarose gel electrophoresis of DNA

- To pour a gel, agarose powder is mixed with electrophoresis buffer to the desired concentration (0.8%), then heated in a microwave oven until completely melted. After cooling the solution to about 60oC, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature. - After the gel has solidified, the comb is removed. The gel, still in its plastic tray, is inserted into the electrophoresis chamber and just covered with buffer. - Samples containg plasmid DNA mixed with loading buffer are then pipeted into the sample wells.

- Set up the electrophoresis apparatus and run at 50 ~ 100 V - The distance DNA has migrated in the gel can be judged by visually mornitoring migration of the tracking dyes. - To visualize DNA, The gel containing DNA is stained by ethidium bromide, and then the gel is observed on a UV transilluminator.

D/RESULT AND DISCUSSION

SAMPLE 9 SAMPLE 1 made by the instructor

1 8

2 9

3 4 10

7
1 2 3

Electrophoresis separate DNA molecules, not only according to their molecular weight, but also according to their shape and topological properties: (Ty mu ca ng m nhn xt nh)

E/CONCLUSION
After the experiement we learn more about the visible form of plasmids and an important technique for DNA extraction Nowadays, with recommbination techniques, and Zinc finger nucleases, plasmids have been enabled the creation of a new generation of isogenic human disease models. The success of some strategies of gene therapy depends on the efficient

insertion of therapeutic genes at the appropriate chromosomal target sites within the human genome, without causing cell injury, oncogenic mutations (cancer) or an immune response. Plasmid vectors are one of many approaches that could be used for this purpose Overall, Plasmid DNA extraction is one of the most basically important technique we need to know