Minipreparation of plasmid from bacterial cells Practical
- 1. Practical #4 Minipreparation of Plasmid from bacterialcells A plasmid is a small, Circular and extrachromosomal double stranded DNA. The structure and function of a bacterial cell is directed by the genetic material contained within the chromosomal DNA. In som cases plasmids are generally not essential for the survival of the host bacterium. Although not essential, plasmids contribute significantly to bacterial genetic diversity and plasticity by encoding functions that might not be specified by the bacterial chromosomal DNA. Plasmids specify traits that allow the host to persist in environments that would otherwise be either lethal or restrictive for growth. Plasmids have been pivotal to modern recombinant DNA technology as a tool in gene-cloning and as a vehicle for gene-expression. Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from bacteria. It is based on the alkaline lysis method. The extracted plasmid DNA resulting from performing a miniprep is itself often called a "miniprep". Minipreps are used in the process of molecular cloning to analyze bacterial clones. A typical plasmid DNA yield of a miniprep is 50 to 100 µg depending on the cell strain. Alkaline lysis is a method used in molecular biology, to isolate plasmid DNA or other cell components such as proteins by breaking the cells open. Bacteria containing the plasmid of interest is first grown, and then allowed to lyse with an alkaline lysis buffer consisting of a detergent sodium dodecyl sulfate (SDS) and a strong base sodium hydroxide. The detergent cleaves the phospholipid bilayer of membrane and the alkali denatures the proteins which are involved in maintaining the structure of the cell membrane. Through a series of steps involving agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris is removed and the plasmid is isolated and purified. It involves the following main steps: i. Growth of Bacterial cells. ii. Suspension Of cells iii. Lysis of cells iv. Neutralization v. Washing Requirements: 1. Growth medium LB medium,1% Tryptone,0.5% yeast extract, 200 mM NaCl 2. Resuspension Solution(I) 50mM glucose,10 mM EDTA,25 mM Tris (pH 8.0). Store at 40 °C.
- 2. 3. Lysis solution ( II) 0.2N NaOH,1% SDS. Store at room temperature 4. Neutralizing solution (III) 3M potassium acetate at ph 6. For 100 ml solution, 60 ml 5 M potassium acetate (49.07 g potassium acetate in 100 ml H2O) 11.5 ml glacial acetate and 28.5 ml H2O, store at room temperature. 5. TE 1 mM EDTA, 10 mM Tris-HCl (pH 8.0) Procedure: i. Grow the bacterial cultures on LB medium with appropriate antibiotics at 37◦Con Shaking. ii. Transfer the bacterial cultures to 1.5 ml Eppendorf tube and centrifuge at 14000 rpm for 1.5 min. iii. Discard the supernatant and add 100 μl of resuspension solution (I) and keep it in room temperatute for 5 mins. iv. Add 100 μl of lysis solution (II) and mix by gently inverting the tube 5-6 times. The solution should quickly turn transparent and become more viscous indicating bacterial lysis has taken place. v. Add 150 μl of neutralizing solution (P3 buffer) and mix by inverting the tubes several times. At this point bacterial chromosomal DNA is usually seen as a white precipitate. vi. Centrifuge the tubes at high speed for 10 min. vii. Carefully transfer the supernatant (try to not disturb the white precipitate) to a new labeled 1.5-ml eppendorf tube with a 1 ml pipette. viii. Add 2.5-3 volume of 200-proof cold ethanol/Isopropanol(stores at -20 °C) to each tube and mix by inverting the tubes a few times. ix. Centrifuge plasmid DNA precipitate (transparency pellet) at high speed for 10 min. x. Discard the supernatant and remove the remaining liquid as much as possible by leaving the tube upside-down on a piece of paper towel, then keep the tubes in a tube holder and air dry for 10-20 min. To dry faster, keep tubes at 37 °C heat blocker. DNA precipitate turns white when dry. xi. Resuspend the DNA pellet with 50 μl TE. Completely dissolve the pellet by pipetting solution several times.