isolation of plasmid DNA from bacteria cell

The isolation of plasmid DNA from bacterial cell using an alkaline lysis is a well-estabilished method. Bacteria with plasmid is cultured in media with antibiotics to a high cell density, harvested, and then lysed with a SDS/NaOH solution. Rapid acidification using concentrated potassium acetate causes the precipitation of protein and chromosomal DNA. Plasmid DNA which is supercoiled remains in solution and can be captures on a silica spin column. The plasmid DNA is washed with a ethanol solution and then eluted in water or TE buffer.

Discard the supernatant and to even invert the tube and wipe the lip with a Kim-wipe or Q-tip.

Resuspend the cells in buffer (often Tris) and EDTA. EDTA chelates divalent metals (primarily magnesium and calcium). Removal of these cations destabilizes the cell membrane. It also inhibits DNases. Glucose should also be added to maintain osmolarity and prevent the buffer from bursting the cells.

Lyse the cells with sodium hydroxide (NaOH) and SDS. This highly alkaline solution gave rise to the name of this technique. Mix this by gentle inversion and incubate on ice for five minutes (but not for longer time).

Renature the plasmid DNA and get rid of the garbage. Add potassium acetate (KAc),

Precipitate the plasmid DNA by alcohol precipitation (ethanol or isopropanol) and a salt (such as ammonium acetate, lithium chloride, sodium chloride or sodium acetate) and spin this down. DNA is negatively charged, so adding a salt masks the charges and allows DNA to precipitate.

Rinse the pellet—plasmid DNA—in ice-cold 70% EtOH and air-dry for about 10 minutes to allow the EtOH to evaporate.

Resuspend DNA pellet in buffer (often Tris) and EDTA plus RNases to cleave any remaining RNA.

Editor: Ankita Added on: 2021-03-07 18:04:37 Total View:360