Molecular Cloning: Competent Cells, Ligation, and PCR

Posted on Jan 11, 2026 in Biochemistry

Preparation of Competent Cells

Aim: To prepare E. coli TOP10 cells capable of taking up foreign DNA (plasmid) using the calcium chloride method.

Core Principle

Calcium Chloride (CaCl₂) neutralizes the negative charges on the cell membrane (phospholipids) and the DNA backbone, reducing electrostatic repulsion. This, followed by a heat shock, creates transient pores for DNA entry.

Key Terms

  • Competence: A cell’s ability to take up extracellular DNA.
  • Transformation: The process of importing foreign DNA.

Critical Step

Harvesting cells at mid-log phase (OD₆₀₀ = 0.4 – 0.5). This is when cells are healthiest and most receptive to becoming competent.

Procedure

  1. Grow a culture from a single colony.
  2. Dilute into fresh media and monitor growth until OD₆₀₀ ~0.5.
  3. Pellet cells and keep everything ice-cold.
  4. Resuspend in ice-cold 0.1M CaCl₂ and incubate on ice for 30 min.
  5. Re-pellet and resuspend in a small volume of CaCl₂ with Glycerol.
  6. Aliquot and flash-freeze for storage at -80°C.

Glycerol prevents ice crystal formation during freezing, preserving cell viability. Always use sterile technique.

Types of Competence

Natural Competence

Genetically predisposed bacteria (e.g., Streptococcus pneumoniae).

Artificial Competence

Induced via chemical (CaCl₂) or electrical (electroporation) methods.

Chemical Competence Details

  • CaCl₂ neutralizes negative charges.
  • Heat shock creates temporary pores.
  • Other chemicals: DMSO, MgCl₂, PEG, RbCl.

Electroporation

Uses an electric pulse to create membrane pores. Reversible (up to 1 kV) vs. Irreversible (up to 3 kV).

DNA Ligation

Joining DNA fragments via phosphodiester bond.

Aim

To join the digested insert (λDNA) and the linearized vector (pUC18) using T4 DNA Ligase.

Principle

T4 DNA Ligase catalyzes the formation of a phosphodiester bond between the 3′-hydroxyl end of one DNA fragment and the 5′-phosphate end of another. This process requires ATP as an energy source.

End Types

  • Sticky Ends: Complementary single-stranded overhangs that ligate efficiently.
  • Blunt Ends: No overhangs; ligation is less efficient.

Rationale for Using Two Enzymes

Using two different enzymes (EcoRI & HindIII) creates non-compatible sticky ends on the vector. This prevents the vector from re-circularizing without an insert and ensures the insert is cloned in the correct orientation.

Standard Reaction Mix

  • Vector DNA (100ng/µl): 9 µl
  • Insert DNA (100ng/µl): 3 µl
  • 5X Ligase Buffer: 4 µl
  • T4 DNA Ligase: 1 µl
  • Sterile H₂O: 3 µl
  • Total Volume: 20 µl

Incubation at 16°C for 2-4 hours (optimal for sticky-end ligation, balancing enzyme activity and end stability).

Note: Aliquot the ligase buffer to avoid degrading the ATP from repeated freeze-thaw cycles. Lower temperatures favor better ligation. Maintain pH 7.6-8.0. Add PEG for efficiency. Restriction enzymes reduce background.

Transformation

Aim

To introduce the ligated plasmid product into competent TOP10 cells and screen for recombinant clones.

Principle

Changing a cell’s genotype by introducing foreign DNA. Cells that take up the pUC18 plasmid (which has an AmpU gene) survive on ampicillin plates.

Key Screening Method: Blue-White Screening

  • pUC18 has a LacZα gene with a Multiple Cloning Site (MCS).
  • Functional LacZα: Cleaves X-gal (with IPTG inducer) → Blue colonies (vector without insert).
  • Disrupted LacZα (Insert in MCS): Cannot cleave X-gal → White colonies (recombinant plasmid).

Transformation Steps (Heat-Shock)

  1. Thaw competent cells on ice.
  2. Add DNA, incubate on ice for 30 min.
  3. Heat-Shock at 42°C for 45 seconds.
  4. Recovery: Add LB broth, incubate at 37°C for 1 hour with shaking.
  5. Plate on LB with antibiotics + Amp + IPTG /X-gal blue/white screening plates.
  6. Spread using a hockey stick or glass spreader; incubate at 37°C overnight.

Essential Controls

  • Negative Control: Cells + no DNA → No growth (confirms antibiotic is working; checks backbone colony formation).
  • Positive Control: Cells + intact plasmid → Many blue/white colonies (confirms transformation worked; checks viability).
  • Experimental: Cells + recombinant DNA → Measures transformation efficiency.

The recovery period is crucial; it allows the bacteria to express the ampicillin resistance gene before being exposed to the antibiotic.

Polymerase Chain Reaction (PCR)

Aim

To amplify the inserted λDNA fragment within the pUC18 vector to confirm its presence.

Principle

A thermal cycling reaction that uses a DNA polymerase to exponentially amplify a specific DNA sequence defined by two primers.

Components

  • Template DNA: The putative recombinant plasmid.
  • Primers: M13 Forward & Reverse (bind outside the MCS in pUC18).
  • Taq DNA Polymerase: Heat-stable enzyme.
  • dNTPs: Nucleotide building blocks.
  • Buffer: Provides optimal pH and Mg²⁺ (cofactor for Taq).

The PCR Cycle

  1. Denaturation: ~95°C – Melts DNA into single strands.
  2. Annealing: ~50-65°C – Primers bind to their complementary sequences.
  3. Extension: ~72°C – Taq polymerase synthesizes the new DNA strand.

Standard Reaction Mix

  • Template DNA: 5 µl
  • Forward & Reverse Primer (10mM): 0.5 µl each
  • dNTPs: 0.5 µl
  • Taq Polymerase: 0.2 µl
  • 10X Buffer: 2 µl
  • Sterile H₂O: 11.3 µl
  • Total Volume: 20 µl

The success of this experiment confirms the presence of the insert. If the insert is present, the M13 primers will produce a PCR product of a specific, predictable size.