How to Make Plasmids?

Making plasmids involves inserting a desired DNA fragment into a circular DNA molecule (the plasmid vector). The most common method is traditional cloning using restriction enzymes and DNA ligase. PCR-based methods offer alternative approaches.

Here's a breakdown of the plasmid construction process:

1. Preparing the DNA Insert

The "insert" is the DNA sequence you want to clone into the plasmid.

• Source: The insert can come from various sources, including genomic DNA, cDNA, or a synthetic gene.
• Amplification (Optional): If the starting amount of your insert DNA is low, you can amplify it using Polymerase Chain Reaction (PCR). This also allows you to add restriction enzyme sites to the ends of the insert.
• Restriction Enzyme Digestion: The insert DNA is digested with one or two restriction enzymes. Choose enzymes that flank the DNA sequence you want to clone and that are compatible with the restriction sites on your plasmid vector. This creates "sticky ends" or "blunt ends" that can be ligated to the vector.

2. Preparing the Plasmid Vector

The plasmid vector is the circular DNA molecule that will carry the insert.

• Choosing a Vector: Select a plasmid vector appropriate for your application. Consider factors like:
  • Origin of Replication: Determines the plasmid's copy number in the host cell.
  • Selectable Marker: Usually an antibiotic resistance gene, used to select for cells containing the plasmid.
  • Multiple Cloning Site (MCS): A region containing multiple unique restriction enzyme sites where you can insert your DNA.
• Restriction Enzyme Digestion: The plasmid vector is also digested with the same restriction enzyme(s) used for the insert. This opens up the circular plasmid, creating compatible ends for ligation.
• Dephosphorylation (Optional): Treating the linearized vector with alkaline phosphatase removes the 5' phosphate groups, preventing self-ligation and increasing the efficiency of insert ligation.

3. Ligation

Ligation joins the insert and the vector.

• Mixing: Combine the digested insert and vector DNA in a tube with DNA ligase and a ligation buffer. The ratio of insert to vector DNA can be optimized to maximize the number of successful ligations. A typical molar ratio is 3:1 (insert:vector).
• Incubation: Incubate the mixture at a temperature and time recommended by the ligase manufacturer (often room temperature or 16°C overnight). DNA ligase catalyzes the formation of phosphodiester bonds, joining the insert and vector DNA.

4. Transformation

Transformation introduces the ligated plasmid into competent bacterial cells.

• Competent Cells: Use competent cells, which have been treated to increase their permeability to DNA. Common methods for creating competent cells include chemical treatment (e.g., using calcium chloride) or electroporation.
• Transformation Method: Mix the ligation reaction with the competent cells and introduce the DNA. Common methods include:
  • Heat Shock: Briefly heat the cells, causing them to take up the DNA.
  • Electroporation: Apply a brief electrical pulse, creating temporary pores in the cell membrane through which DNA can enter.
• Recovery: After transformation, incubate the cells in a nutrient-rich medium (e.g., LB broth) to allow them to recover and express the antibiotic resistance gene.

5. Selection

Selection identifies bacterial cells containing the plasmid.

• Plating: Plate the transformed cells onto a selective agar plate containing the antibiotic corresponding to the resistance gene on the plasmid.
• Incubation: Incubate the plates at 37°C overnight. Only cells containing the plasmid (and therefore the antibiotic resistance gene) will grow and form colonies.

6. Screening

Screening identifies colonies containing the plasmid with the correct insert.

• Colony PCR: Pick individual colonies and perform PCR using primers specific to the insert. This allows you to quickly screen for colonies containing the desired insert.
• Restriction Digestion: Isolate plasmid DNA from selected colonies and digest with restriction enzymes that cut within the insert. The resulting fragment sizes can be compared to the expected sizes to verify the presence and orientation of the insert.
• Sequencing: Sequence the plasmid DNA to confirm the correct sequence of the insert and the junctions between the insert and the vector.

Example: Cloning a Gene into a Plasmid

1. Target Gene: You want to clone the GFP (Green Fluorescent Protein) gene into a plasmid.
2. PCR Amplification: Amplify the GFP gene using PCR, adding EcoRI and HindIII restriction sites at the ends of the amplified product.
3. Restriction Digestion: Digest both the amplified GFP gene and the plasmid vector with EcoRI and HindIII.
4. Ligation: Ligate the digested GFP gene and plasmid vector together using DNA ligase.
5. Transformation: Transform competent E. coli cells with the ligation mixture.
6. Selection: Plate the transformed cells on LB agar plates containing ampicillin (assuming the plasmid carries an ampicillin resistance gene).
7. Screening: Perform colony PCR or restriction digestion to identify colonies containing the GFP gene insert. Sequence-verify positive clones.

Alternatives to Traditional Cloning

While restriction enzyme digestion and ligation are standard, other methods exist, including:

• Gibson Assembly: Allows for the joining of multiple DNA fragments in a single reaction, without the need for restriction enzymes. Requires overlapping sequences at the ends of the fragments.
• TOPO Cloning: Utilizes the enzyme topoisomerase to directly ligate PCR products into a vector.
• Gateway Cloning: Uses site-specific recombination to transfer DNA fragments between vectors.