Study Guide for Lab Exam II

Basic Parts of the Microscope

• Components: Ocular lens, objective lenses (4x, 10x, 40x, 100x).
  • Total Magnification: Calculated by multiplying the power of the objective lens by 10 (Total Magnification = Objective Lens Magnification x 10).

Preparing a Wet Mount Slide

• Steps:
  • Start with a clean microscope slide.
  • Add a drop of water in the center of the slide.
  • Place the specimen into the liquid of water.
  • Place a coverslip onto the slide.

Determining Cell Density of Tetrahymena Sample

• Steps:
  • Place a drop of the culture on a clean slide and cover with a coverslip.
  • Observe cells under phase contrast to examine swimming motion.
  • Remove excess water by touching a piece of filter paper to the end of the coverslip.
• Calculation: Multiply the average count per square by 10^4.
  • Example: If you count 20 cells in a 0.1 mL sample, the concentration is 20 cells/0.1 mL = 200 cells/mL = 2x10^4 cells/mL.

Gel Electrophoresis

• Matrix: Agarose gel is used to separate DNA based on size.
• Migration: DNA migrates towards the positive electrode because it has a negatively charged sugar-phosphate backbone.
• Process: Gel electrophoresis applies an electrical field across a porous gel matrix to separate DNA molecules based on their size and charge.

Determining Size of DNA Fragments

• Technique: Use gel electrophoresis to measure the size of DNA fragments.
  • Range: Size range for accurate determination is from 100 to 10,000 base pairs.
• Method: Loading digested DNA into gel wells and applying an electrical current allows separation based on size.

Restriction Enzymes

• Definition: Enzymes produced by bacteria that cut DNA at specific sequences to protect against viral DNA.
• Usage: Essential tools in molecular biology for creating specific DNA fragments.
• Source: Scientists obtain them from bacteria.

Cleavage of DNA

• Plasmid vs Linear DNA:
  • Cleaving a plasmid at one site generates linear DNA fragments (2 fragments).
  • Cleaving a linear DNA at one site creates two smaller linear fragments.

Visualizing DNA Fragments from Gel Electrophoresis

• Staining Method: Staining with fluorescent dyes such as ethidium bromide and exposing to UV light allows visualization of separated DNA fragments.

Conditions for Cell Growth

• Optimum Growth Conditions:
  • Temperature: Generally between 18°C to 57°C, most mammalian cells require around 36°C.
  • CO2 levels and humidity are essential for growth.

Necessity of Sterile Techniques

• Purpose: To prevent contamination by microorganisms during cell culture.
  • Practices:
  1. Handwashing
  2. Wearing gloves
  3. Setting up a sterile environment

Experiments in Cell Culture Lab

• Cell Line Used: Defined specific mammalian cell line.
• Determining Live vs Dead Cells: Utilized hemocytometry combined with trypan blue dye (live cells exclude the dye, dead cells take it up).

Research Project

• Focus: Conducted research on cyclin E.
• Gene Sequence Database: Identified through specified databases.
• BLAST Program Usage: To compare the sequence against other biological sequences and evaluate evolutionary relationships.

Evaluating Evolutionary Relationships

• Method: Compare DNA or protein sequences across organisms and construct phylogenetic trees based on similarities and differences.