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
Sample Retrieval:
Students will retrieve their tube containing the DNA sample.
Bead Handling:
Each student receives a PCR tube with beads; avoid opening until ready.
Students must label their tubes with initials and numbers.
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.
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:
Initial Denaturation:
Temperature set at 94 °C to denature the DNA strands (separate the double-stranded DNA).
Denaturing Phase:
Lower temperature allows DNA primers to attach to the single strands.
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.