8. Microscopy & Lab Techniques

PCR (Polymerase chain reaction) general

-"Copy machine" -Provides a way to make copies of a small sample of DNA

If the objective lens is 4x and the eyepieces lens is 4x, what is the total magnification?

-4 * 4 = 16x is the total magnification -Total magnification is objective lens * eyepiece lens

Genome library

-A way to store all the DNA of an organism's genome -

Staining

-Adding color to cells in order to better see certain structures -Staining is usually associated with cells that have been killed either through fixation step or the staining process itself

Transgenic animals

-Allows researchers to study the function of gene sequences -Gene sequence will be taken from one type of organism and inserted into another -Can create transgenic animals for production of medications

DNA microarray

-Allows us to determine which genes are being expressed in a cell or tissue 1. Isolate mRNA from a cell 2. Using reverse transcriptase, synthesize cDNA from mRNA 3. To distinguish between the 2 pools of cDNA, strands are fluorescencently labeled 4. DNA added to DNA array and they hybridize with an complementary DNA in the array 5. Wherever binding has occurred is indicated by the fluoresce of the DAN

Pulse chase

-Allows us to track the movement of a radioactive molecule, like a protein, through a cell 1-Pulse phase: radioactively labeled amino acids is added to a cell during a short window. And they will be incorporated into the gene products (proteins) 2-Chase phase: non-radioactive amino acid is added to the cell and so no more radioactively labeled amino acids are incorporated into the gene products 3-Next researcher can track the radioactive proteins movement through the cell -Chase phase prevents every protein a cell makes from being labeled, which makes it easier for researchers to identify the proteins of interests

Dark field microscopy (optical microscopy)

-Allows us to view unstained samples of live cells -This is because there is more contrast between the sample and the background -Light image produced on darker background -Dark field patch spot (blue bar in image) blocks most light from illuminator and so the only light you see is light reflected from structures in the samples your looking at

Scanning electron microscopy (SEM)

-Allows us to visualize high resolution 3D images of the sample surface of dehydrated samples -Scans sample with a beam of electrons > electrons interact with surface atoms > produces a 3D image of the sample's surface -High resolution

Western blotting

-Allows you to detect the presence and amount of our protein of interest from a pool of proteins -Achieved using sodium dodecyl sulfate polyacrylamide gel (SDS Page) 1. Proteins are denatured and given neg charge by sodium dodecyl sulfate 2. Proteins travel through gel electrophoresis from negative cathode to positive anode. Proteins are separated by size and charge 3. Proteins are transferred to a sheet (membrane) that is then exposed to radio labeled primary and secondary antibodies that bind epitopes of target proteins 4. Protein bands detected by specific antibodies are visualized

Strep

-Bacteria grows in a chain

Metal Coating (electron microscopes)

-Bacteria solution fixed on slide -Then a heavy metal salt solution stain is placed on top of the slide

Staph

-Bacteria that grows in clusters

Spirilla

-Bacteria with a spiral or curved/cork-screw shape -Do NOT form chains

Transmission electron microscopy (TEM)

-Beam of electrons passed through a very thin section of a sample to produce a 3d image of internal structures -High resolution -Kills sample

Bacterial growth curve

-Can be used go describe the growth pattern of a given culture of cells -Graph is in log # of viable bacteria vs time -3 phases: lag phase, exponential phase, stationary phase, death phase

Cell fractionation and proteins

-Can separate proteins based on solubility -Insoluble proteins will end up in the pellet -Soluble proteins will remain dissolved in the supernatant

Compound microscope (optical microscope)

-Can view simple, one-cell thick, live cells -Have more than one lens (objective lens and ocular lens) -2D image, low resolution

Cell fractionation

-Cell contents are separated into their fraction by centrifugation

Electrical resistance

-Cells show great electrical resistance (they don't conduct electricity) -So the # of cells can be estimated by observing the flow of electricity

Diaphragm

-Controls the amount of light passing through the sample

Rheostat

-Controls the intensity of the light

Electron tomography

-Creates 3D image of sample's internal structures -Achieved by using a bunch of TEM images together -Not a type of microscopy

Recombinant DNA and restriction enzymes

-DNA fragments from different sources are joined together -Restriction enzymes create the fragments of DNA -They tend to cut DNA at palindromic sequences to produce sticky or blunt ends

Restriction fragment length polymorphism (RFLPs)

-Differences among individual's lengths of DNA fragments cut by restriction enzyme -If 2 people have differences in their DNA sequences at particular restriction sites, then the restriction enzymes will cut their DNA into fragments of different lengths -Genomic fingerprints as every individual will have different length RFLPs, except for identical twins

Stereo microscopes (optical microscope)

-Dissection microscope -Only offers low magnification to observe the surface of live specimens -Low resolution

Blunt ends

-Do not have unpair nucleotides -Makes them harder to hybridize with other blunt ends

Pellet vs. Supernatant

-During centrifugation, heavier components will sediment to the bottom, becoming pellets or precipitate -Supernatant is the liquid on top of the pellet

Electroporation

-Electricity is applied to cells -Makes cells competent

Which provides a higher resolution image, electron microscopy or optical microscopy?

-Electron microscopy because the wavelength of an electron is smaller than that of light

Can electron microscopy or optical microscopy be used to view living cells?

-Electron microscopy cannot not view living cells; cells must be fixed, stained, and killed -Optical microscopy can be used to view living cells

Southern blotting

-Enables us to determine the presence of certain nucleotide sequences in a sample of DNA 1. Cleave DNA into fragments 2. Run gel electrophoresis on DNA fragments to separate fragments based on charge and size 3. Transfer gel to filter to visualize fragments 4. Expose filter to radio-labeled piece of DNA that is the complement of our gene of interests 5. Radio-labeled DNA will anneal to the complement gene of interest 6. Expose filter to X ray to visualize results (if we see radio-labeled DNA, the gene of interests was present in our sample)

Fixation

-Fixing a cell on a microscope slide -Getting cells to stick on the slide, such that they are preserved in its most life like state -Prevents post-death decay/degradation

Lag phase

-Flat phase -No active growth yet, bacteria are adjusting/adapting to conditions -Constant population size -A

Condenser

-Gathers and focuses light on the specimen being viewed

Short tandem repeats (STRs)

-Group of nucleotides that repeat again and again in a stretch of DNA

Primary antibody (western blot)

-Has a target antigen -Will selectively bind a target protein

Stage clips

-Holds the slide in place on the stage

Hemocytometer

-Instrument used for counting cells -Hemocytometer is a gridded slide -Grid is used to manually count the # of cells in a known area

Secondary antibody

-Is attached to an indicator that glows a certain color -It also has a primary antigen and will bind to the primary antibody

Knockout mice

-Knockout mice have a selected gene of interest "knocked out" -Then changes are observed between wild type and knockout mice

Illuminator

-Light source for the microscope

Centrifuge/centrifugation

-Machine that spins in a circular path at very high speeds -This separates the cell comments through their mass. density, and/or shape

Fluorescence Recovery After Photobleaching (FRAP)

-Measuring lateral mobility of living membrane proteins -Membrane proteins are tagged with a fluorescence (we measure baseline fluorescence) -A selected area is bleached with a laser, this removes fluorescence -Over time, photo bleached molecules are replaced by unbleached molecules -Quick recovery time = very motile

Next generation sequencing

-Method for DNA sequencing -Quicker and cheaper than Sanger sequencing

Death phase

-More cells dying than being produced due to depletion of nutrient and space and the build up of toxins in the culture -D

Mechanical stage knobs

-Move the slide from side to side -Doesn't help you focus the image, just helps you explore what's on the slide

Karyotyping

-Observation of chromosomes under a light microscope -Performed at metaphase of mitosis -Can be used to diagnosis chromosomal abnormalities

Dideoxy chain termination (Sanger sequencing)

-Older and more established method for DNA sequencing

Diplo

-Pairs of bacteria

Log (exponential) phase

-Plenty of nutrient and space for bacteria to grow -B

Fluorescence Lifetime Imaging Microscopy (FLIM)

-Provides a quantitative measure of the concentration of various ions, molecules, and gases in a cell -Accomplished by treating cells with light and measuring their fluorescence lifetime -Fluorescence lifetime refers to the amount of time it takes for an excited molecule to release all of its flurosence

CRISPR-Cas 9 (Clustered Regularly Interspace Palindromic Repeats)

-RNA guide sequence is complementary to a sequence in target the DNA -Cas9 binds to RNA guide sequence and this complex binds to DNA -Cas9-RNA complex cuts DNA and an alternative DNA sequence to be placed in that spot -Form of genome editing; cut and paste tool

Coarse focus

-Raises and lowers the stage vertically -Does so in large increments

Fine focus

-Raises and lowers the stage vertically, but in small increments -Moves the stage slightly to sharpen the image

Stationary phase

-Rate of cell growth equals rate of cell death caused by depleted nutrients and O2, and build up of toxins and waste products -Constant population size -C

Nose piece

-Rotates and holds the objective lens

Northern blotting

-Same as southern blotting but helps identify fragments of a known RNA sequence using RNA probes

Bacterial cloning (general)

-Scientist clone eukaryotic gene products in prokaryotic cells -Can use bacteria cell machinery to make gene products

Density centrifugation

-Separates all the cell components of the original homogenate in a single centrifugation cycle -Separates all sediments into layers based on density

Optical microscopy

-Shining light on sample -Then the light that reflects off the sample passes through a series of lenses which magnify the object -Can be used to observe living cells

Significance of Dolly the sheep

-Showed somatic cells do contain the entire genome -And showed that multipotent cells can be reverted to their totipotent state (can divide and produce an entire organism)

Cryo-scanning electron microscopy (cry-SEM)

-Specific type of SEM where the sample is frozen in liquid nitrogen instead of being dehydrated -Kills sample -High resolution -Sample presented in a more natural form, but freezing can cause artifacts (damage caused in specimen preparation that can be confused with being apart of the specimen's structure)

Coccus

-Spherical bacteria

Stage

-Supports the slide being viewed

Fluorescence microscopy/confocal laser scanning

-Tag certain structures with fluorescent marker, then use laser light to scan specimen -2D image is displayed digitally -Living sample, can look at specific parts of the cells (i.e. chromosomes during mitosis) -Fluorescence can cause artifacts

Reproductive cloning

-Taking a stomata cell from an animal and producing a genetic copy from that cell -No genetic variation created -Somatic cell must be converted from its multipotent state (very differentiated state) to a totipotent state (can divide and produce an entire organism)

Objective lens

-The lens on a light microscope that is closest to the stage -Have different ranges of magnification

What is negative of compound microscopes?

-They provide poor contrast and samples are best viewed when fixed or stained (but fixing and staining will KILL a living cell)

Down syndrome

-Trisomy 21 -Extra copy of the 21st chromosome

Sticky ends

-Unpaired nucleotides on one strand -Makes it easy for complementary sticky ends to hybridize -Complementary sticky ends (of donor DNA and vector) are made from the same restriction enzyme

DNA fingerprinting

-Used in paternity and forensic cases -Identifies individuals through aspects of unique DNA, like RFLPs and STRs -Can match an individual to a sample

Enzyme-linked immunosorbent assay (ELISA)

-Used to determine a specific antigen exists in a person -Helps diagnosis diseases like HIV 1. Take blood from person 2. Antibodies from blood are placed into a microtiter plate 3. If any antibodies bind to the antigen being tested for, there will be a color change in microtiter plate -Color change indicates that the specific antigen is present in the person. Otherwise, why would they have an antibody for that antigen

Colony forming units (CFUs)

-Used to estimate the number of viable bacteria cells in a sample -Viable = ability to multiply via binary fission -Estimate the # of cells present based on their ability to give rise to colonies under specific conditions

Gel electrophoresis

-Used to separate DNA fragments based on their charge and size -Can be used to determine genetic similarities and differences -Applies an electrified to gel -Gel used: agarose = polysaccharide -Negative cathode at top and positive anode at bottom -DNA is negatively charged and moves towards the positive charge -DNA is cut into fragments using restriction enzymes -Longer DNA fragments pieces move slower -Shorter DNA fragments pieces move faster

Bright field microscopes (optical/compound microscope); what it is and its disadvantages

-Uses a bright light to illuminate the sample -You are viewing a darker image on a lighter background -Low contrast makes it difficult to view the sample. So staining has to be used but this kills the sample

Electron microscopy

-Uses electrons instead of light -It bombards a sample with electrons and the electrons that bounce off the sample pass through a series of magnetic fields and ultimately land on a screen -Image is view indirectly using a computer

Phase contrast mircoscopes

-Uses light phase changes and contrast to produce a 2D image of thin samples -Allow visualization of thin sample containing live cells. Can also view the internal structures -Cells do not need to be fixed, stained, tagged because these microscopes have tremendous amounts of contrast -Ineffective on thick samples -Can create halo effect around sample edges

Single nucleotide polymorphism (SNPs)

-Variation in a DNA sequence occurring when a single nucleotide in a genome is altered -These are position in the genome where some individuals have one nucleotide and others have a different

Color change method

-Vectors containing a gene that makes cells blue will be used -Restriction enzymes that cut blue gene will also be used -If the target gene inserts into the blue gene, blue gene will be inactivated and the cell will be white -If target gene does not insert into blue gene, blue gene will be activated and cell remain blue

Vector

-Vehicle to artificially carry foreign genetic material into another cell -Piece of DNA that can be taken up by competent cells

Heat shock

-Way to make bacteria cells competent -Solution containing CaCl2, bacteria, and vectors is heated which creates holes in the bacterial cell membranes enabling vectors to enter and transformation to occur

Heat fixation

1. Cells are placed on one side the microscope slide 2. The underside of that same slide will be passed over a Bunsen burner flame -The heat causes the cell to be glue on the slide -Heat also preserves the cells because the heat causes cell processes to stop (kills them)

Differential centrifugation

1. Cells must be split open so that comments can be separated (homogenization) 2. In homogenate mixture, nucleus will pellet first because it is the most dense. Everything else remains in supernatant 3. Supernatant will then be poured into a new centrifuge tube so the next most dense organelle can pellet 4. Repeated to gradually isolate the cell comments so they can be studied

PCR steps

1. Denature (95 C) - using heat to separate the 2 single strands of DNA 2. Anneal (65 C) - cooling DNA to allow DNA primers to bind on separate complementary strands 3. Extend (70 C) - Taq DNA polymerase elongates each strand 4. Repeat to get more DNA strands

2 types of automated cell counting

1. Electrical resistance 2. Flow cytometry

1. If you want to visualize organelles what microscope would you use? 2. If you want to look at different cells, what microscope would you use?

1. Electron microscope; better magnification 2. Light microscope

Steps of bacterial cloning (explain antibiotic resistance method and color change method)

1. Processed mRNA (just extorns) for the eukaryotic gene of interests is isolated 2. mRNA is treated with reverse transcriptase to make cDNA 3. Restriction enzyme and DNA ligase allow cDNA to be incorporated into a plasmid 4. Plasmid is taking up by competent bacterial cell and bacterial cell becomes transformed 5. We find gene of interest by using antibiotic resistance or color change methods

Microscope resolution

Ability of a lens to separate or distinguish small objects that are close together

Flow cytometry

Cells pass through a very narrow tube and can be counted via detection by a laser beam

Eye piece lens (ocular lens)

Contains a lens that magnifies 10x

DNA sequencing

Determining the exact order of the base pairs in a segment of DNA

Fluorescence

Emission of photons from a particle that has absorbed light

Microscope magnification

Makes an image larger

Most dense to least dense cell organelles

Nuclei > mitochondria/chloroplast > ER fragments > ransoms

Palindromic sequence

Nucleic acid sequence: read the same 5'-3' as complementary 5'-3'

Bacillus

Rod shaped bacteria

Genomics

Study of an organism's genome