Lab Techniques PDF

# Microscopies and Lab Techniques ## Microscopes ### **Fixation** - Preserves the shape - Adheres them to the slide ### Staining - Increases visibility of the specimen by adding color ## Types of Microscopes ### Comparison Table | Microscope | Type | Views | Mechanics | Other | |---|---|---|-...

# Microscopies and Lab Techniques ## Microscopes ### **Fixation** - Preserves the shape - Adheres them to the slide ### Staining - Increases visibility of the specimen by adding color ## Types of Microscopes ### Comparison Table | Microscope | Type | Views | Mechanics | Other | |---|---|---|---|---| | Stereo Microscope| OPTICAL | The exterior of a specimen | Low Magnification, 2-D image | Used in dissections | | Compound Microscope | OPTICAL | Simple, single layered live cells | Multiple lenses | Staining and fixation must be performed to avoid inferior contrast | | Bright Field Microscope | OPTICAL | Simple, single layered live cells | Identical to compound microscopes, but with an additional bright light | N/A | | Phase Contrast Microscope | OPTICAL | Live cells that are arranged to be flat and thin | 2-D image; the annular ring refracts light and leads to high contrast | High phase shifts, Halo Effect | | Fluorescence Microscope | OPTICAL | Variety of parts in a cell are viewed, by (exposing specimen to UV, blue, or violet light) | Dichroic mirror only allows few wavelengths to pass | Clarity is decreased by distortions and artifacts | | Confocal Laser Scanning Microscopy | OPTICAL | Fluorescent objects | A laser beam is focused directly on the sample to reduce artifacts | Samples should be illuminated longer due to decreased intensity because of the focusing of the laser beam | | Dark Field Microscopy | OPTICAL | Live cells that are not stained | Image formed by light refracted by sample. | Final image is a bright image against a dark background, very high contrast | | Scanning Electron Microscopy | ELECTRON | Surface of a sample that has been dehydrated and stained with heavy metals. | Final image is a very clear 3-D depiction of the sample. | Aka SEM | | Cryo-scanning Electron Microscopy | ELECTRON | Surface of a sample that has been frozen in liquid N₂ | Final image is a very clear 3-D depiction of the sample. | Aka Cryo-SEM, Expensive technique and manufactures artifacts. | | Transmission Electron Microscopy (TEM) | ELECTRON | Interior of the cell | Final image is a very clear 2-D depiction of the sample. | Electron tomography can be used to blend TEM images to create a 3-D image of the interior structures. | ### Optical Microscope vs Electron Microscope #### Optical Microscope - For living and non living cells - Low resolution - Larger organelles: Nucleus, cytoplasm, chloroplast, cell wall - **Directly** #### Electron Microscope - For nonliving cells - High resolution - Smaller organelles: Nucleolus, ribosomes, RER, SER, centrioles, mitochondria, lysosomes. - Electrons bounce and pass through a magnetic field. - **Indirectly ** # Biological Lab Technique ## Counting Cells - Hemocytometer: Counts the number of cells in a gridded area. - Colony Forming Units: Estimates the number of cells in a colony (ic=10). - Automated Cell Counting: Laser that detects the number of cells. ## Cell Fractionation - Used to separate cellular components based on size and density. - The bottom of the pellet has larger/denser organelles than the top. - Has 3 steps: Extraction, homogenization, and centrifugation. ## Centrifugation - Separates particles based on size, shape, and density - **Differential Centrifugation:** Based on size, larger particles (nucleus) sediment faster than smaller particles (ribosomes). - **Density Centrifugation:** The bottom layer is the densest, and the uppermost layer is the least dense. **Order of Elution / First to Last / Most Dense to Least ** - Nucleus - Chloroplast - Mitochondria - Lysosome - Peroxisome - Microsomes - Ribosomes ## Karyotyping - Counting the number of chromosomes using a light microscope during metaphase (mitosis) to check for abnormalities. - Down syndrome, Turner's syndrome, and Klinefelter's syndrome can be diagnosed using this technique. ## DNA Sequencing - Determines the order of nucleotides. - Includes the older method (Sanger Sequencing), and the new method (Next Generation Sequencing) ## *(SNP) Diff in the Genome Sequence* - **Single Nucleotide Polymorphisms (SNP):** Used to directly find the gene causing a disease. ## Recombinant DNA - Restriction enzymes cut DNA at palindromic sequences (Sticky ends are unpaired nucleotides; blunt ends paired nucleotides) - **Restriction Fragment Length Polymorphisms (RFLP):** Used to compare and contrast DNA between individuals ## DNA Fingerprinting - Short tandem repeats and RFLP are used to identify individuals. ## Polymerase Chain Reaction (PCR) - Creates millions of copies of DNA in 3 steps: (resulting strand only has human DNA). - **Denaturation (95°C):** Heat is used to separate strands of DNA. - **Annealing (65°C):** DNA primers adhere to the template DNA. - **Elongation(70°C):** Tag Polymerase (DNA polymerase) adds nucleotides to the 3' ends - $2^n$ = # of cycles > copies - $2^{x2}$ = # of strands after x cycles. ## Bacterial Cloning - Prokaryotic cells are injected with eukaryotic genes. ## Reverse Transcriptase - cDNA, Bacteria, Plasmid ## Plasmid - Carry antibiotic resistance genes; Small circular DNA that can replicate independently ## Gel Electrophoresis - Separates macromolecules (DNA, RNA, Proteins) based on size and charge. - **Top:** Consist of negative particles - **Bottom:** Has positively charged molecules - **Negative samples** go to the **+** side - **Positive samples** go to the **-** side ## Southern Blotting - Detects specific pieces of DNA ## Northern Blotting - Detects specific pieces of RNA ## Western Blotting - Detects specific pieces of proteins. - Uses primary and secondary antibodies. ## ELISA (Enzyme Linked Immunosorbent Assay) - Used to detect if a person has a specific antigen present. ## Pulse Chase Experiments - Allows us to see how proteins move through a cell over time. - RER GA Secretory Vesicles > Plasma membrane - **Pulse:** Uses radioactive amino acids to label proteins. - **Chase:** Prevents the division of the labelled proteins. ## Hershey-Chase Experiment - Proteins are labelled with radioactive sulfur. - DNA is labelled with radioactive phosphorus. - **Showed that DNA was the transforming molecule** ## Genome Annotation - Identifies the location of genes, which are coding and which are noncoding regions ## SDS (Sodium Dodecyl Sulfate) - Used on proteins to denature non covalent bonds, add charges, detangles proteins. ## SDS Page - Separates proteins based on mass. ## Genomics - Field that studies genes and their interaction within a genome ## Genomic Library - Stores the DNA of an organisms genome ## DNA Microarrays - Determines which genes are expressed (active). ## Transgenic Animals - A gene is transferred from one organism to another to determine its function ## Reproductive Cloning - Using a somatic cell to produce a copy of an organism's genome. - **Exp:** Dolly the sheep ## Totipotent - Evolve to become a complete organism. ## Pluripotent - Evolve into either ectoderm, mesoderm, or endoderm ## Multipotent - Evolve into 1 or 2 of the germ layers, but not all 3, most differentiated only produce 1 type of cell. ## Chromatography - Separates particles based on solubility ## Retention Factor - $ Rp = \frac{D_{1}}{D_{2}} $ - $D_{1}$ = Distance traveled - $D_{2}$ = Distance solvent ## Fluorescence Recovery After Photobleaching (FRAP) - Cells are bleached and when they divide, the daughter cells are not bleached. - See how molecules move in a cell ## Fluorescence Lifetime Imaging Microscopy (FLIM) - Cells are illuminated and their lifetime is measured. - Determines concentration. ## Knockout Mouse - A specific gene is removed to see its function. ## Bacteria - **Diplo:** Pairs - **Strep:** Chain - **Staph:** Grape-like clusters ## Coccus - Spherical ## Bacillus - Rod-like ## Spirilla - Spiral ## Bacterial Growth Curve Steps of Growth Pattern: - **Lag Phase:** Bacteria are adapting to growth conditions. - **Log (Exponential Phase)**: Number of cells and rate of growth doubles. - **Stationary Phase:** Results from growth-limiting factors. - **Death Phase:** Bacteria die due to lack of nutrients, environment temperature, etc. Decline in viable cell count. ## Transformation - When bacteria uptake genetic material from the environment. - **Competent Bacteria:** Are the ones that can do transformation. ## Transduction - Viruses transfer bacterial DNA ## Conjugation - Gene is transferred using a cytoplasmic bridge.