DNA

DNA Extraction: A method for isolating and purifying DNA by using physical and chemical methods from a sample; during this process, DNA is separated from cell membranes, proteins, and other cellular components.

The Three Steps of DNA extraction: cell lysis, precipitation and purification

Cell Lysis: Barriers such as the cell membrane and the membranes of the nucleus break open in order to expose DNA. This step involves mechanical disruption and the use of enzymes and detergent to dissolve cellular proteins and free DNA.

Precipitation: DNA is separated from cellular debris. This step involves the removal of DNA- and RNA-associated proteins by protease and RNase. Sodium ions are used to neutralize the negative charge in DNA molecules, making them less water soluble. Alcohol causes precipitation of DNA from the solution because DNA does not dissolve in alcohol.

Purification: The DNA is rinsed with alcohol, which removes all the remaining cellular debris and unwanted material

Polymerase Chain Reaction (PCR): A technique for selectively amplifying a specific segment of DNA.

The Three Steps of PCR: Denaturation, Annealing, and Extension

Denaturation of a double stranded DNA (dsDNA) template at approximately 92–95°C, which separates the sample into single stranded DNA (ssDNA) components.

Annealing of primers by allowing the sample to cool to approximately 50–65°C; this allows the primer to hydrogen bond to each existing ssDNA.

Extension of dsDNA molecules at approximately 72°C by adding nucleotides complementary to the template strand.

Taq DNA polymerase: Isolated from Thermus aquaticus, extends the primers by adding nucleotides complementary to the template strand. Taq DNA polymerase is most effective at 750 C. (temperature in the hot springs from which the enzyme was isolated).

Tools to preform DNA extraction:

  • Container with cracked or crushed ice

  • Microcentrifuge

  • 6 microcentrifuge tubes (1.5 mL)

  • Micropipettes and tips (2-1000 µL)

  • Permanent marker

  • 2 plastic pestles

  • Heating Blocks at 65°C and 57 °C

  • Vortexer

  • Microcentrifuge tube rack

Tools to preform PCR:

  • Micropipettes and tips (1-100 µL)

  • Permanent marker

  • Thermal cycler

  • Container with cracked or crushed ice

  • Microcentrifuge tube rack