Isolation of plasmid

Introduction

Plasmids are extrachromosomal double-stranded circular DNA that self-replicates and are widely used for cloning foreign DNA into the bacteria as host strain. The basic properties of plasmids:

Different forms of plasmids

The bacterial plasmid is a double-stranded circular DNA that exists in 3 different forms.

• Supercoiled DNA (acquire supercoiled configuration)
• Open circular DNA (one of the strands has nick -OC)
• Closed circular DNA (both strands of circular double strands are intact – covalently closed circles -CCC

Features of different plasmids

There are minimum molecular components to assemble bacterial plasmid to perform the function of the vector are as follows:

1. Origin of replication – it has an independent origin of replication to provide a replication start site to make more copies.
2. Selection marker- A selection marker in the form of either an antibiotic resistance gene or an enzymatic gene is essential to express genes present on plasmid DNA.
3. Promoter – promoter is needed for the express gene present on plasmid DNA in the host organism.

Materials and Instruments

1. E. coli cells culture containing plasmid to extract DNA.
2. Solution I (glucose, TrisHCl pH 8.0, EDTA), glucose increases osmotic pressure outside the cell.
3. Solution II (NaOH and SDS), NaOH raptures the cell wall and SDS raptures the lipid membrane
4. Solution III (potassium acetate, glacial acetic acid.), to lower down the pH.
5. Phenol: Chloroform: Isoamyl alcohol -Mix Phenol (pH 8.0), chloroform and Isoamyl alcohol in the ratio of 25:24:1, mix by vortexing.
6. Absolute Ethanol
7. Agarose and Buffers for Electrophoresis

Steps in isolation in bacterial plasmid

Step 1: resuspension and lysis of bacterial cells

Each Bacterial cell contains chromosomal DNA, plasmid DNA, and cellular proteins.

1. Bacterial culture is collected by centrifugation at the bottom discard the supernatant and invert the tube on a dry paper towel to dry the pellet.
2. Resuspended the bacterial pellet in the solution I containing glucose, TrisHCl pH 8.0, EDTA pH 8.0. Break the cell pellet by vortexing for 2 minutes.
3. Incubate the Bacterial pellet on ice for 5mins. This step will partially lyse the bacteria by hypotonic osmosis and release cellular content.

Step 2: Alkaline Lysis

1. Bacterial cells are treated with solution II containing NaOH and SDS. Mix by inversion. Incubate the tube on ice for 5 minutes. This step will completely lyse the cells and denature DNA (both chromosomal and plasmid DNA) and protein.

Step 3: Renaturation

1. Add ice-cold Solution III. Mix by inversion and incubate on ice for 5mins. In this step, denatured DNA is renatured with solution III containing potassium acetate, and glacial acetic acid. In this step, small DNA (plasmid) renature back quickly whereas chromosomal DNA remains denatured.
2. Centrifuge at 12000g for 5 minutes and transfer the clear supernatant to a fresh tube to avoid contamination of white precipitate.
3. Add RNase A to the supernatant. Incubate the tube at 37
   oC for 30min. This step will degrade the RNA present in the sample.

Step 4: Deproteination

1. The resulting supernatant containing plasmid DNA and protein is treated with an equal volume of phenol: chloroform: isoamyl alcohol mixture and mixed by vortexing for 1 minute. This step removes protein from the solution in the precipitate whereas the plasmid remains in the solution.
2. Centrifuge at 12000g for 5 minutes and transfer the upper clear supernatant to a fresh tube.
3. Add an equal volume of Phenol: Chloroform: Isoamyl alcohol (25:24:1) and mix by vortexing.
4. Centrifuge at 12000g for 5 minutes and transfer the upper clear supernatant to a fresh tube.

Step 5: Precipitation

1. Add 2 volumes of absolute ethanol and mix by vortexing to precipitate the plasmid DNA.
2. Centrifuge at 12000g for 5 minutes and discard the supernatant. Wash the DNA pellet with the 70% ethanol.
3. Centrifuge at 12000g for 5 minutes and discard the supernatant.
4. Air dry the pellet and dissolve the pellet in the sterile molecular biology grade water or TE Buffer (Tris pH 8.0 containing 10mM EDTA).
5. Analyze the DNA on the 0.8% agarose gel.
   Observation: Analysis of plasmid DNA on the agarose gel gives 3 bands corresponding to the 3 different forms of DNA.