Laboratory Techniques in DNA Manipulation - Week 5: DNA Cloning and PCR

Laboratory Techniques in DNA Manipulation

Week 5: DNA Cloning and PCR Overview
  • Instructor: Dr. Harisree Paramel Nair
  • Email: hp29@aru.ac.uk
  • Today’s focus is on PCR amplification of GST and preparation for ligation into pBluescript.
Learning Outcomes
  • Health and Safety: Evaluate lab health and safety considerations.
  • Laboratory Techniques: Perform DNA techniques and analyze experimental data.
  • Bioinformatics: Manipulate and analyze bioinformatic data to answer experimental questions.
  • Mathematical Applications: Apply knowledge of molarity and scientific notation in laboratory analysis.
Today's Experimental Procedures
  • PCR Amplification: Amplifying the GST gene using identified primers.
  • Validation and Purification: Check PCR success and purify the product.
  • Restriction Digestion: Digest PCR product with restriction enzymes.
Protocol: PCR Mixture
  • Components:
    • Template DNA (GST)
    • Oligonucleotide Primers (specific for GST)
    • Buffer and Cofactors (MgCl2)
    • dNTPs
    • Taq DNA Polymerase
  • Process:
    • Combine constituents in a PCR tube.
    • Transfer to a thermocycler for thermocycling (50 mins).
PCR Reaction Mixture
  • Ice Bucket Setup:
    • Distilled deionized water: 11.5µL
    • Forward Primer (F): 1.25µL
    • Reverse Primer (R): 1.25µL
    • Template DNA: 1µL
    • Master Mix: 12.5µL
  • Total Volume: 25µL
Post-PCR Steps
  • Visualize Results: Check for amplified DNA of expected size using bioinformatics predictions.
Agarose Gel Electrophoresis
  • Principle: DNA is negatively charged and migrates to positive electrodes in an electric field.
  • Size Separation: Smaller fragments travel faster through the gel than larger ones.
Preparing Agarose Gel
  • Materials: 1g agarose, 100mL TAE buffer (40mM Tris, 20mM acetic acid, 1mM EDTA).
  • Steps:
    • Weigh agarose and add TAE buffer.
    • Microwave to dissolve, then add 6µL of Gel Red.
    • Pour into gel tray after cooling slightly and insert a comb for well formation.
Running Agarose Gel
  • Electrophoresis Setup:
    • Load DNA samples mixed with loading dye into wells.
    • Use a DNA ladder for size reference.
    • Fill tank with TAE buffer and run at 150V for 30-40 minutes.
Loading Samples
  • Sample Preparation: Mix PCR product with loading dye to ensure sinking.
  • Loading Technique: Carefully pipette samples into the wells without piercing through.
Restriction Digestion Setup
  • Prepare Tubes for pBluescript and PCR Product:
    • Tube A (PCR Product): 15µL PCR product, 5µL restriction buffer, 28µL ddH2O, 1µL BamHI, 1µL HindIII.
    • Tube B (pBluescript): 15µL pBluescript, 5µL restriction buffer, 28µL ddH2O, 1µL BamHI, 1µL HindIII.
  • Incubation: Store at 37°C for one hour, then at 4°C.
Health and Safety Considerations
  • Use PPE: Lab coats, gloves, and eye protection.
  • Substances:
    • Ethylenediaminetetraacetic acid (EDTA): Serious eye irritation; harmful if inhaled.
    • Acetic acid: Flammable; causes severe skin burns.
Practical Competencies
  • Obtain sign-off sheet and submit upon completion of lab tasks for bonus marks.
Clean-up Protocol
  • Clean and organize workspace; dispose of waste properly, and disinfect surfaces with Chemgene.