PCR & Gel Electrophoresis

PCR Protocol Essentials

• Core workflow: Get reagents; Prepare the mix; Set up conditions; Run/Analyze the gel; Interpret negative results.

Gel Electrophoresis: Purpose and Mechanism

• Purpose: separate DNA molecules by size using an agarose gel under an electric field.

• DNA charge: negatively charged due to the phosphate backbone; migrates toward the positive electrode.

• Matrix effect: agarose gel acts as a mesh; smaller fragments move faster/farther than larger ones.

Gel Running Parameters and Matrix

• Gel concentration (resolution):

  • Typical: $1.0\%\ \text{agarose}$

  • Higher: $2-3\%\ \text{agarose}$ to improve resolution of similar-sized fragments

  • Lower: $0.7-1\%\ \text{agarose}$ for faster runs

• Voltage: typically $200\ \text{V}$; higher voltages increase heating; monitor temperature.

• Buffer: running in a gel box with $0.25X\ \text{TAE}$ buffer to reduce heating; TAE = Tris, acetate, EDTA.

• Run mnemonic: "Run to Red" (DNA runs toward the red (positive) end).

Loading and Visualization Essentials

• Loading dye purposes:

  • Visible color to track loading

  • Denser than buffer to pull sample into wells

  • Dye itself is negatively charged and migrates toward the positive end

  • May contain a fluorescent stain for visualization

• DNA visualization: the DNA stain is excited by $364\ \text{nm}$ light and fluoresces at $454\ \text{nm}$.

• Safer alternatives: Ethidium bromide is mutagenic; class uses non-toxic fluorescent loading dye.

• DNA ladder: run a DNA ladder with known sizes to estimate fragment size (example sizes: $100\ \text{bp}, 200\ \text{bp}, 500\ \text{bp}, 1000\ \text{bp}$).

PV92 ALU Insertion Interpretation (PCR Product Sizes)

• Amplicon sizes:

  • $641\ \text{bp}$ if no ALU insertion

  • $941\ \text{bp}$ if ALU insertion present

• Genotypes:

  • $+/+$ homozygous insertion

  • $-/-$ homozygous no insertion

  • $+/-$ heterozygous

• Band interpretation:

  • Smaller band (641 bp) often more intense due to amplification efficiency

  • Heterozygotes can be misread as homozygous if the larger band is faint

• Heteroduplexes in $+/-$ samples: larger bands around $-1100\ \text{bp}$ and $-1700\ \text{bp}$ due to duplexes between alleles

• Primer-dimers: small bottom bands, more common with little template or contamination

Gel Loading Order (Common Layout)

• Left side:

  • Lane 1: DNA ladder

  • Lane 2: $+/+$ homozygous control

  • Lane 3: $+/-$ heterozygous control

  • Lane 4: $-/-$ homozygous control

  • Lanes 5-8: your group samples

• Right side: additional lanes (as per protocol)

Practical Lab Concepts and Tips

• Why gels are run: to visualize fragment sizes and infer genotypes from band patterns.

• Temperature and gel quality: avoid overheating; use appropriate buffer concentration and voltage.

• Safety and ethics:

  • Ethidium bromide is mutagenic; non-toxic dyes are preferred for visualization.

  • Follow institutional guidelines for disposal.

Troubleshooting and Common Explanations

• Empty lanes (no amplification):

  • Inadequate collection of cheek cells; need visible cell pellet after centrifugation

  • Too many cells saturating the InstaGene matrix

  • InstaGene matrix not transferred or beads not suspended

  • Carryover of matrix into PCR inhibiting the reaction

• Heterozygote interpretation caveats:

  • Competition during amplification can make the smaller band appear stronger; heterozygotes may look homozygous if the larger band is faint

• Gel artifacts:

  • Heteroduplex bands (1100-1700 bp) in heterozygotes

  • Primer-dimer bands at the bottom of the gel

Quick Reference: Setup and Measurements

• Typical loading setup (example): mix sample with loading dye; load ladder; run at $200\ \text{V}$; monitor toward red; keep gel in $0.25X\ \text{TAE}$ buffer.

• PV92 interpretation basics: eligible bands at $641\ \text{bp}$ and $941\ \text{bp}$ indicate absence/presence of ALU insertion, respectively.