PLASMID DNA ISOLATION FROM E.coli (TENS Method)
INSTRUMENTS: Autoclave, Centrifuge, Refrigerator,Shaker incubator, Micro pippetts and tips, appendorf tubes.
PRINCIPLE: The isolation procedure is based on the release of soluble molecular weight DNA from disrupted cell wall and membrane
...
, dissociation of nuclear protein by denaturation and proteolysis and separation of DNA from macromolecule. Bacteria are lysed with a solution containing SDS and NaOH; during this step chromosomal as well as plasmid DNA are denatured. Subsequent neutralization with sodium acetate allow only the covalently closed plasmid DNA to reanneal and to stay solubilized. Most of the chromosomal DNA and protein precipitate in a complex formed with sodium and SDS, which is removed by centrifugation. The plasmid DNA is concentrated from the supernatant by ethanol precipitation.MATERIALS REQUIRED:
1. TENS solution:
• 10 mM Tris (pH to 7.5)
• 1 mM ethylenediaminetetraacetic acid (pH to 8.0 to dissolve)
• 0.1 N sodium hydroxide
• 0.5 % sodium dodecyl sulfate
2. 3 M Sodium acetate, pH 5.2
3. Pre-chilled (at -20 degrees C) 100 % ethanol
4. 70 % Ethanol
5. Distilled water
6. Overnight bacterial culture
PROCEDURE:
1. A volume of1.5 ml of overnight bacterial culture was spinned for 30-60 seconds in a micro centrifuge. (observation:bacteria form a pellet at the bottom of the tube.)
2. The supernatant was discarded, leaving 50-100 ul in the tube.
3. The bacteria pellet was vortexed to resuspend completely.
4. A volume of 300 ul of TENS solution was added.
5. It was vortexed for 5 seconds to mix.(observation:The contents of the tube should become slimy)
6. A volume of 150 ul of the sodium acetate was added.
7. It was vortexed for 5 seconds to mix.
8. The above was spinned for 2 minutes in a microcentrifuge. (observation: a white pellet, containing bacterial debris,is formed at the bottom of the tube.)
9. Supernatant was transferred to a fresh tube.
10.A volume of 0.9 ml of Pre-chilled 100 % ethanol was added.
11.It was spinned for 5 minutes in a microcentrifuge. (observation: a white pellet, containing plasmid DNA and bacterial RNA, is formed at the bottom of the tube.)
12.The supernatant was discarded and 1 ml of 70 % ethanol was added.
13.The ethanol was discarded and another 1 ml of 70 % ethanol was added to wash.
14.As much liquid was withdrawn & discarded (ethanol) as possible then the pellet was air-dried
15.The pellet was resuspended in 30ul of distilled water and kept at 4 degrees C or -20 degrees C.
RESULT: The plasmid DNA was isolated from given bacterial culture.
INFERENCE: TBE buffer has EDTA which might interfere later in restriction digestion, hence the resuspended pellet was stored in distilled water. To check the purity of obtained DNA, further analysis was done by agarose gel electrophoresis.
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