Question

2) Please explain how to purify plasmid DNA from bacterial cells.

Answer 1

Plasmid DNA is small, circular DNA present in the bacterial cells. They are separate from the chromosomal DNA and carries genes which offer certain advantages to the host cells carrying them. The replication of the plasmid DNA is independent of the chromosomal DNA replication. Plasmid DNA can be easily isolated from the prokaryotic cell and is used in various processes like cloning, sequencing, PCR etc.

Isolation of plasmid DNA from bacterial cells is generally done using the alkaline lysis method. The steps in the process is given below.

1. Take a culture of bacteria carrying the desired plasmid in a sterile tube.

2. Centrifuge the tube and remove the medium. The pellet contains the bacterial cells.

3. The pellet is re-suspended in a resuspension (50 mM glucose, 25 mM Tris-Cl, 10 mM EDTA). This step helps denature DNA, destabilises the cell membrane and deactivates DNases which degrade the DNA.

4. Add the lysis buffer (0.2N NaOH, 1% SDS) to this solution. Mix gently and incubate on ice for few minutes. SDS (Sodium dodecyl sulfate) causes cell membrane lysis and releases the plasmid and chromosomal DNA from the cell. NaOH (Sodium hydroxide) causes DNA denaturation.

5. Add neutralising solution (3M potassium acetate) to the mix. This step causes precipitation of the SDS-protein complex. It also neutralises the solution which leads to renaturation of DNA. the SDS-protein complexes and the chromosomal DNA precipitates as they are heavy. However, plasmid DNA being smaller in size, remains in the supernatant.

6. centrifuge the tube and collect the supernatant containing the plasmid DNA into a fresh tube.

7. alcohol precipitation method is used to precipitate the plasmid DNA from the supernatant.

alcohol precipitation method

1. Add equal volumes of phenol and chloroform to the supernatant and mix well. Centrifuge and remove the aqueous layer on top to a fresh tube.

2. to this add, 2x volume of ethanol and mix well. The plasmid DNA gets precipitated and can be collected as the pellet after centrifugation.

3. wash with 70% isopropanol to remove excess salt present in the plasmid DNA precipitate. To achieve this step, add 70% isopropanaol to the pellet and mix gently. Centrifuge thetube and remove the supernatant.

4. leave the tubes open to remove all traces of isopropanol from the plasmid DNA pellet.

5. The plasmid DNA can be resuspended in Tris-EDTA buffer and stored at -200C.