Chpater 12 ~ BIO101 Lab

PCR Ingredients

• DNA sample (two per group)
• Polymerase: An enzyme that makes the DNA molecule by adding nucleotides.
• Nucleotides: Adenine, thymine, cytosine, and guanine.
• Primers: Short strands of DNA (or RNA in the body) needed to start DNA replication.

PCR Steps

• Denaturation: Heat is used to separate the two strands of DNA.
• Annealing: Primers attach to the DNA strands.
• Extending: Polymerase adds nucleotides to the primer, creating a new DNA strand.

ALU Insertion

• A piece of DNA (transposon) on chromosome 16.
• ALU negative: Chromosome 16 without the ALU insertion (416 base pairs between primers).
• ALU positive: Chromosome 16 with the ALU insertion (731 base pairs between primers).
• Individuals can be homozygous negative, homozygous positive, or heterozygous.
• Does not affect phenotype.

Gel Electrophoresis

• Used to separate DNA fragments by size.
• Smaller fragments travel farther in the gel.
• Homozygous negative: DNA travels far.
• Homozygous positive: DNA doesn't travel as far.
• Heterozygous: Results in both bands.

Pipettes

• P10: Smallest pipette.
• P100: Medium-sized pipette.
• P1000: Largest pipette.

Lab Procedure

• Collect DNA sample and put in cold water.
• Centrifuge.
• Transfer to a tube of Chelex (to disable coenzymes).
• Heat in heat block.
• Vortex and centrifuge.
• Transfer supernatant to a new tube and put on ice.
• Add primer to the PCR bead.
• Add DNA, centrifuge (using adapter tubes), and put initials on the tube.

Foam Activity

• Hands-on activity to illustrate PCR; replace video activity.