10/30/25

Pipetting Technique

  • Everyone has a pipette to practice with.
  • Instructions on using the pipette:
    • Press the button to begin suctioning the liquid.
    • Stop pressing the button when you feel pushback; this indicates the liquid is at the first stop.
  • Pipettes have two stops:
    • The first stop represents the set volume (e.g., settings of 100-1,000, 2-20, or 10-100 microliters).
  • The pipetting task involves transferring liquid into designated spots on paper circles (e.g., 10, 20, 40, 80 microliters).
  • Participants are encouraged to practice pipetting accurately.

Polymerase Chain Reaction (PCR) Overview

  • Introduction to PCR following the collection of cheek cell DNA samples.
  • PCR is defined as a polymerase chain reaction used to amplify DNA.
  • Purpose of PCR:
    • To increase the number of DNA copies for further analysis.
    • Essential for observing genetic material.
  • Key components of a PCR reaction:
    • DNA samples from the previous session.
    • A bead containing:
    • Taq polymerase: enzyme used in PCR.
    • Nucleotides: building blocks for DNA synthesis.
    • Buffer: solution that maintains optimal pH and ionic environment for the reaction.
  • The components are mixed using pipettes; practice is emphasized before proceeding.
  • PCR is facilitated by a thermocycler which cycles through heating and cooling.

Procedure for Setting Up PCR Reaction

  1. Sample Retrieval:
    • Students will retrieve their tube containing the DNA sample.
  2. Bead Handling:
    • Each student receives a PCR tube with beads; avoid opening until ready.
    • Students must label their tubes with initials and numbers.
  3. Pipetting DNA Sample:
    • Set pipette to 10 microliters (100 setting for a 2-20 pipette).
    • Transfer 10 microliters of the DNA sample into the PCR tube.
    • Mix the content by pipetting up and down to dissolve the bead into a liquid form.
  4. Master Mix Addition:
    • After initial mixing, students will wait for instructions.
    • Each student will subsequently pipette 15 microliters of a master mix into their PCR tube. The master mix contains:
      • Water.
      • Primers: short sequences of nucleotides that initiate DNA synthesis.
    • Check pipette settings before dispensing the master mix.

Thermocycler Functionality

  • The thermocycler is crucial for the PCR process, engaging in repeated temperature changes to facilitate DNA amplification.
  • Steps cycled in the thermocycler:
    1. Initial Denaturation:
    • Temperature set at 94 °C to denature the DNA strands (separate the double-stranded DNA).
    1. Denaturing Phase:
    • Lower temperature allows DNA primers to attach to the single strands.
    1. Extension Phase:
    • Medium temperature enables Taq polymerase to synthesize new DNA strands.
  • This cycle occurs 35 times.
  • Finally, samples are stored at 4 °C.

Course Structure and Exam Information

  • General class structure is supported by PowerPoint presentations.
  • Information reiterated multiple times during lectures is critical and likely to appear on exams.
  • Important to note equations, such as that for photosynthesis, which may be crucial for tests.
  • Test schedule includes various assessments:
    • Third unit test planned just before Thanksgiving in November, includes a cheat sheet (specifics like paper size can vary).

Laboratory Communication and Engaging the Class

  • Students are encouraged to engage actively with questions and discussions about lab processes.
  • The importance of lab accuracy is emphasized, using examples from previous experiences (e.g., discussing COVID DNA testing and its amplification process).
  • The instructor fosters an open environment for students to express difficulties or concerns that may arise during practical labs.

General Remarks

  • Students should ensure proper handling and cleanliness in lab preparations to prevent contamination of their samples.