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Plasmid Miniprep This procedure is used to extract plasmid DNA from bacterial cell suspensions and is based on the alkaline lysis procedure developed by Birnboim and Doly Nucleic Acids Research 71513 1979 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 plasmids and RNA molecules The cells are lysed under alkaline conditions which denatures both nucleic acids and proteins and 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 correctly and stay in solution effectively separating them from chromosomal DNA and proteins Procedure Note The procedure below is used to make duplicate minipreps This provides balanced tubes for the centrifuge as well as twice as much product when you are finished 1 Gently swirl the contents of the culture tube to resuspend the cells 2 Label two 15 ml tubes and pipette 1000 uL of the cell suspension into each tube 3 Close the caps and place the tubes in a centrifuge remember to balance the centrifuge by putting the tubes opposite one another and spin at maximum speed for 20 s This part of the procedure collects the bacterial cells which are suspended in the liquid medium into a cell pellet at the bottom of the tube 4 Withdraw and discard the supernatant using a pipette being careful not to disturb the cell pellet Discard the supernatant in a waste container 5 Add 100 uL of Buffer 1 50 mM Tris-HCl 10 mM EDTA 100 ugmL RNase A pH 80 to each tube and resuspend the cells by vortexing Its very important that the cell suspension is homogenous and no
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