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Materials: 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 3 M Sodium acetate, pH 5.2 Pre-chilled (at -20 degrees C) 100 % ethanol 70 % Ethanol Distilled water Overnight bacterial culture (LAB 5)Supplies: Micropipetter and tips Vortex mixer Microcentrifuge and tubesProcedures: Spin 1.5 ml of overnight bacterial culture for 30-60 seconds in a microcentrifuge. (observation: bacteria form a pellet at the bottom of the tube.) Decant supernatant, leaving 50-100 ul in the tube. Vortex to resuspend the bacteria pellet completely. Add 300 ul of TENS solution. Vortex for 5 seconds to mix. (observation: the contents of the tube should become slimy.) Add 150 ul of the sodium acetate. Vortex for 5 seconds to mix. Spin for 2 minutes in a microcentrifuge. (observation: a white pellet, containing bacterial debris, is formed at the bottom of the tube.) Transfer supernatant to a fresh tube. Add 0.9 ml of pre-chilled 100 % ethanol. Spin for 5 minutes in a microcentrifuge. (observation: a white pellet, containing plasmid DNA and bacterial RNA, is formed at the bottom of the tube.) Discard supernatant and add 1 ml of 70 % ethanol. Discard the ethanol and add another 1 ml of 70 % ethanol. Withdraw and discard as much liquid (ethanol) as possible then air-dry the pellet. Resuspend the pellet in 30 ul of distilled water and keep at 4 degrees C or -20 degrees C.Results: Plasmid DNA is now ready for estimation of DNA concentration (LAB 4) followed by restriction digest (LAB 3). Typical yield is 2-3 ug.
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