Microscopy and Lab Techniques DAT CH 8

Objective

part of the microscope that magnifies the image

fixation

sticks cells to the slide preserving them in their most life like state- kills them

Staining

adds color or contrast to the cells, making the cell structures easier to see- kills them

Optical microscopy

cells are viewed directly, light shines on samples which is magnified via the lenses- can be used to observe living cells

Electron microscopy

cells are viewed indirectly, electrons are fired at the samples, then bounce off and pass through magnetic fields. Used to view smaller objects, cells must be fixed using metal coated staining- kills the cell

Phase contrast microscopes

View thin samples of live, unstained cells with high contrast

Fluorescence microscopy

live cells tagged with fluorophores (fluorescent chemicals). Can visualize cellular components

Scanning Electron Microscopy (SEM)

High resolution 3d images of the surface of a dehydrated sample

Transmission Electron Microscopy (TEM)

High resolution 2d images of a samples internal structures

Bacterial growth curve

Lag, Exponential, Stationary, Death

Lag Phase

Adaption prior to cell division in Bacterial Cell growth curve

Exponential (Log) Phase

Rapid doubling in Bacterial Cell growth curve

Stationary Phase

Growth rate= death rate in Bacterial Cell growth curve

Death phase

decline due to lack of food/other variable

Differential centrifugation

used to separate cellular components based on their size and density by subjecting them to centrifugal forces in a series of steps

exonuclease

enzyme that cleaves nucleotides from the polynucleotide chain at the end of the chain, results in sticky ends

endonuclease

enzyme that cleaves nucleotides from the polynucleotide chain, results in sticky or blunt ends

Restriction enzymes

specialized endonucleases that mostly cut DNA at a specific palindromic sequence to produce either sticky or blunt ends to create recombinant DNA

Karyotyping

observing chromosomes under a light microscope during metaphase

CRISPR

used to edit specific genomic regions of interest by adding or deleting target sequences of DNA. Used in gene therapy

DNA fingerprinting

identifies individuals through analyzing specific regions of noncoding DNA, fragmented with restriction enzymes. Used in paternity or forensic

DNA sequencing

determines the order of nucleotides in a DNA strand, Sanger sequencing

Sanger sequencing

A type of DNA sequencing where DNA copies are made by PCR using dnTPS and ddnTPS (lack 3’ OH terminating)

Bacterial cloning

Cloning eukaryotic gene products in prokaryotic cells, used to produce medicine

RNA → Reverse transcriptase → cDNA → Plasmid → DNA ligase→ Competent bacteria transformation → antibiotic resistance or color change

Bacterial cloning protocol

Polymerase Chain Reaction (PCR)

Automated process creating 1+ billion copies of DNA in three steps 1. Denaturation (hot) 2. Primer Annealing (cold) 3. Elongation (warm) with taq polymerase

Gene therapy

process of inseriting genes into cells to treat disease using viral or non viral vectors, naked plasmid DNA or CRISPR

Enzyme Linked Immunosorbent Assay (ELISA)

Determines if a person has a specific antigen. Important to diagnose diseases, Antibodies are placed on a plate with a sample and change color if antigens are present (both bound)

Pulse Chase Experiment

studying gene expression and fate of proteins, pulse- amino acids are radioactively labeled and inserted chase- prevent previously radioactively labeled amino acids from persisting

Gel Electrophoresis

Separates fragments of nucleic acids or proteins by charge and size

Top

Location of negative cathode in gel electrophoresis

Bottom

Location of positively charge anode in gel electrophoresis

Southern Blotting

fragments of known DNA, complementary DNA probes (SNOW DROP)

Northern Blotting

identifies fragments of known RNA using an RNA probe (SNOW DROP)

Western Blotting

Quantifies amount of target protein in a sample using DNS Page and treated with primary and secondary antibodies (indicator and binder) (SNOW DROP)

Genomics

study of all the genes present in organisms genome

Genome annotation

after genome has been sequenced, identifies the location of genes and coding regions in a genome

Genomic Library

stores the DNA of an organisms genome, DNA fragments are incorporated into plasmids and cloned using bacterial cloning

DNA microarrays

contain thousands of DNA probes that in to complementary DNA fragments, allowing researchers to see which genes are expressed. Similar protocol to Bacterial cloning

Immunofluorescence microscopy

Technique that identifies the localization of proteins of interest using fluorophores

Fluorescence Recovery after photobleaching (FRAP)

quantitative measure of how and where biomolecules move in a live cell, bleach and area and graph how quickly unbleached cells move in