Introduction to Laboratory and Scientific Methodology
Introduction to Laboratory and Scientific Methodology
Popular in Course
Popular in Life Science
verified elite notetaker
This 32 page Bundle was uploaded by Michelle Vaday on Saturday April 5, 2014. The Bundle belongs to a course at University of California - Los Angeles taught by a professor in Fall. Since its upload, it has received 228 views.
Reviews for Introduction to Laboratory and Scientific Methodology
Report this Material
What is Karma?
Karma is the currency of StudySoup.
You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!
Date Created: 04/05/14
23L 4514 934 PM 0 Lecture 1 CMIT o beginning information written down write the rest later Neuropsychology studies relationship between brain and behavior can be helpful to determine if individual suffers from cognitive disorder Left side of the brain academic Language Logic Analytical thinking Mathscience language right side of the brain creative creativity spirituality imagination identification of color frontal part of brain abstract reasoning problem solving decision making attention and concentration and behavior control motor cortex movement sensory sensations parietal lobe perception spelling occipital lobe vision temporal lobe hearing language and understanding motor and sensory cortex contralateral right side of brain controls left side of body outer areas of brain higher cortical areas cerebrum cognition language and visual and attention abilities inner areas or sub cortica areas are responsible for basic survival needs wakefulness thirst and breathing coma damage inner areas such as brain stem cognition measured by paper and pencil tests Stroop color word inference color naming word reading hard 9 easy Wordreading fastest overlearned and on one side of the brain left Color naming on right side of the brain so needs to come back to the left Color word inference utilize frontal lobes When required to read a word O Occipital lobes see word Wernicke s area Broca s area where you say the word Naming color Occipital lobes Then go to the other side of the brain where non verba color identifying abilities are Processed back to Wernicke s Broca s area to say words out of lips Should take more time C color word inference most difficult task use frontal lobe used to make decisions tells the brain to go to the other side to identify the color and come back to identify process to name it out loud helps regulate and suppress impulses frontal lobes usually not completely developed until 25 Some individuals do not get enough oxygen or stimulation in frontal lobes to focus and concentrate Stroop can diagnose individuals with ADHD HIV crosses the brain blood barrier One test was Picture Memory interference test PMIT More updated version is the CMIT Ease in administration of measure Larger pool of data Explore more research questions Measure response reaction time CMIT currently used to detect HIV associated neurocognitive disorders among Spanish speaking individuals in LA HAND vs no HAND groups People with no hand score almost perfectly Book Notes 2 important emphases in practicing science 1 They hypotheticodeductive approach senses of steps that leads to a robust conclusion about a problem requires a specific hypothesis 9 prediction of an effect or a difference 2 The falsifactionist procedure involves taking the hypothesis and creating a null hypothesis which will predict no effect or no difference between two or more tested samples Making a generalization based on one or more observations is called an inductive generalization These are constantly open to revision Statistics is divided into 2 types descriptive eg mean and standard deviation with describe the pattern of measurements and inferential statistics eg t test used to assess whether 2 samples are coming from the same population The standard deviation is a measure of variation around the mean T test useful tool that determines the difference between two samples by comparing their means while taking into account their variances The outcome of the t test is the t vaue but this tells us nothing so to determine if our sample groups are different using this number we look at the probability value or the p vaue P vaue of 005 is called the significance level This tells us that we are only 5 likely to have made a mistake by rejecting the null hypothesis If your pvalue is greater than 5 005 you will retain the null hypothesis and conclude that the two groups are not significantly different If however the pvalue is less than 5 you will conclude that the two samples are significantly different In addition to recording right and wrong answers the MIT program measures reaction time TA NOTES Ex hypothesis male and female users perform differently in the picture memory test Nullhypothesis male and female perform the same in picture memory test 0 Pvalue probability that the differences between 2 pops are due to chance The t test assesses whether the means of the two groups are statistically different from each other 0 Lecture 2 Epidemiology and Laboratory Techniques epidemiology is the study of diseases Epidemiologists investigate the sources routes of transmission and patterns of infectious diseases Most commonly used instrument in microbiology is the micropipeter Concepts Disease transmission and preparing a serial dilution AIDS can be traced back to Africa SARS broke out in china Swine flu originated in Mexico and spread worldwide within a year One individual became infected with HIV in Haiti and then brought it to the US HIV then spread around the globe AIDS can remain undetected for years SARS and swine flu are much faster spreading Epidemiology is a field of science which tries to understand the spread of a disease and furthermore by predicting its patterns to prevent its occurrence in the first place H191 virus swine flu pandemic Two scientists looked at this Origin in Mexico Soon first infections in us were registered Then western Europe Asia and eastern Europe Question is why the virus appears earlier in some country than others effective distance on this new map you see locations that are particularly close to each other Simple singular wave map Prediction of location or origin successively placing all airports and checking to see if it could be the starting point of the epidemic It will not determine the first person that transmitted the infection 9 known as patient zero They are the starting point they go undetected Patient 0 of swine flu was a 5yearold boy who lives by a pig farm 3 states of pipetor released state first stop second stop no not go beyond the first stop serial dilution any error will be noticeable if it is too diluted come up with explanation loading an agarose gel solid block of film gel it is submerged in buffer Sample should go in buffer Submerge hover first stop pull out and release Loading a 96 we pate Go to the side then first step Next go to the second stop The pull out the tip and then release the push button To retrieve a sample from a 96well plate Push down to the first stop find the bottom of the well without pushing it shut on the bottom then push the release button slowly and pull out the tip from the well use second stop if you are in an open container but not in the gel Book Notes Epidemiology is the study of diseases and populations at risk HIV infects human T ces by binding to a protein called CCR5 which enables it to enter the cell and being propagating and causes immune cell death 10 of Europeans are naturally immune to HIV because they have a mutated form of CCR5 rendering the HIV virus unable to enter the cells and replicate An assay is a simple test used to qualitatively or quantitatively analyze a sample Agarose gel enables us to separate DNA fragments based on size and then view these fragments under ultraviolet light Proteins functional products of our genes Antibodies small molecules that recognize specific targets are evidence of an immune response We can produce antibodies in the lab that recognize HIV proteins SDS PAGE gel separates proteins based on their size and conformation Separating proteins based on size can help you identify which proteins are present A gel can give us a qualitative assessment of the presence or absence of DNA or protein it does not provide us a good quantitative value The spectrophotometer can accurately determine the concentration of DNA RNA or protein in a sample The spectrophotometer has limits and therefore a concentrated sample must sometimes be diluted to get a more accurate reading 1000 pg 1 ng 1000 p 1 nl 1000 ng 1 microg 1000 n 1 microl 1000 microg 1 milligram 1000 microiter 1 milliliter 1000 miigram 1 gram 1000 miiiter 1 liter 3 pipettors One with volumes ranging from 1001000 microliters p1000 20200 microliter p200 and 220 microliter p20 on p1000 the third number represents 100039s the second is hundreds and the third are the lowest digit tens TA Notes 0 common errors with pipetting pushing to second stop before aspirating 9 over delivery not pushing to second stop when expelling liquid 9 under delivery failure to pause after placing tip in sample and before aspirating sample 9 variability not touching pipette tip gently to the side of tube when expelling sample 9 under delivery Lecture 3 Beta Gaactosidase Activity understanding lac operon and function use protein activity assay to determine enzymatic activity lactase is an enzyme that breaks down lactose into galactose and glucose belongs to class of proteins called beta gaactosidase actose sugar found in milk and all mammals will express lactase at birth but may not express as an adult if you don39t have mutated gene then you are actose intoerant Will produce a lot of gas Lactase has been extensively studied in ecoi uses the lac operon to metabolize lactose Has one chromosome and several plasmids The chromosome is circuar contains 2 regions the lac repressor and lac operon First step in gene expression is transcription DNA 9 RNA Promoter lays 10 bases before gene Where RNA pol starts transcription Following transcription the mRNA is recognized by the ribosome large complex of proteins and RNA and site of protein synthesis Translation RNA into amino acids Lactase is regulated on transcription level as series of genes called an operon Operon series of genes whose expression is coordinated together Lac operon mechanism for ecoi does detect nutrients in its environment Includes 3 structural genes lac Z Y and A also includes regulatory elements Lac P common promoter Also region called the operator Operator is binding site for lac repressor key for regulation of lac operon Lac repressor coded from lac I is not part of operon itself LacZ encodes lactase found in cytosol Breaks down lactose into galactose and glucose used for energy It isomerase lactose into a molecule called allolactose allolactose is an isomer of lactose functions as an indicator of presence of lactose in the cell acY allows lactose to enter from the environment into the bacterium acI DNA binding protein RNA pol will bind to promoter region and transcribe the gene The repressor gene mRNA is translated by ribosomes to repressor protein Repressor binds to operator region blocking the translation of structural gene In absence of lactose there is a minimal level of allolactose and repressor stays bound to the operator As a result the 3 genes Z Y and A are not transcribed to RNA so proteins are not translated either In the presence of lactose some is converted to allolactose then binds to repressor which induces change that eliminates ability to bind to the operator Repressor no longer blocks binding Once the lac genes are expressed lactose is metabolized to glucose and galactose Allolactose is called an inducer of ac operon When lactose is absent lac operon repressed When glucose is absent and lactose is present lac operon is enhanced Many bacteria including ecoi prefer ATP as source of energy If glucose is available bacteria utilize it first before resorting to other energy sources CAP DNA binding protein and has 2 states 0 If CAP is binding small molecule of cAMP cap will change conformation and bind to activator region near promoter 0 If no cAMP cap doesn39t bind to activator region 0 CAP assists RNA pol in binding These depend on glucose levels in the cell When glucose low cAMP is high and CAP can bind to activator expression of lac operon is enhanced utilizes lactose as energy source When glucose high cAMP low unable to bind expression of lac operon low level Uses glucose as energy source Forthelab We have to break open the cells to get to the enzymes Is a substance called IPTG which is molecular analog of allolactose synthetic molecule structurally similar to allolactose but IPTG cannot be broken down over time expression of lac genes maintained at high level known as an inducer cannot be broken by beta gaactosidase ONPG artificial substrate for galactosidase similar in structure to lactose ONPG is colorless but cleavage by B gaactosidase produces yellow compound 420 nm IPTG induces expression of the lac operon gene ONPG acts as a substrate You will break the cells open with chloroform to stop the gene expression at an exact time point Book Notes The enzyme B ga catalyzes the hydrolysis of lactose to galactose and glucose Bacteria growing in glucose no lactose have no need for the enzyme b gal Enzyme activity is measured using a biochemical assay indirectly assesses how much of a given protein enzyme is produced per cell per unit of time A derivative of lactose ONPG is used as a substrate which is a colorless compound Cleavage of ONPG by B ga produces a yellow compound Luria broth provides essential nutrients for bacterial growth Z buffer optimizes the pH of the sample Addition of Na2CO3 stops reaction of B ga and ONPG by changing pH from 70 9 11 B ga is VERY pH sensitive 06 acidic 7 neutral and 814 basic TA Notes 0 lactose use b ga 9 galactose glucose 0 ONPG b ga 9 galactose o nitropheno yellow 0 Students split tasks for cell density assay and b ga assay between 20 and 70 min Lubria broth for cell density spec and bb ga blank for b ga assay Cell density 600 nm and b ga at 420 nm 0 acZ encodes b ga 4514 934 PM Lecture 4 Metabolism Handling godfish formulate predictions about metabolic rates Designing experiment with controls and proposing a testable hypothesis T test to understand p vaues WRITE SECOND LABOROTORY RESEARCH PAPER Metaboism related to energy and enzymes Chemical reactions occur in living organism Metabolism is the totality of these reactions in any given instant Catabolic break down nutrient molecules release energy ATP Anaboic synthesize macromolecules and use up ATP energy Enzymes lower the energy barrier Enzyme will speed up the reaction Warming increases rate of reaction rate but if temp gets too high enzymes will destabilize Enzymes can change activity by expression in different isoforms times and interacting with other molecules or enzymes Poikilotherm fish or reptile has a temp that varies with the temp of its surroundings is an ectotherm Homeotherm organism such as mammal or bird that has a body temp that is constant and independent of temp of its surrounding is an endotherm At all temp the metabolic rate of lizard is slower than the mouse heart rates among species are not at all similar the larger the animal the slower its resting heart the total metabolic rate increases with body mass Metabolic rate of small endotherm is greater than small endotherm The smaller the animal in body mass the larger the relative metabolic rate Smaller animals have a relative larger metabolic rate than larger animals Metabolism in fish relies on 3 things Respiration and nutrition to supply metabolites Osmoregulation for a stable working environment Excretion to get rid of all poisons and waste products produced as sideeffects Cataboism breaking down metabolites to produce active energy Anaboism building new body tissue for growth maintenance and reproduction Metabolic rate can change with a variety of factors Size bigger fish have slower metabolic rates Age young fish grow more but don39t need the reproductive side yet Activity busy fish need a faster rate Condition fish in poor condition need more tissue maintenance Environment temp oxygen salinity affect the rate if everything is normal in fish39s environment it produces energy by oxidation requires constant supply of enough oxygen cold water can hold oxygen better than warm water fish has a certain tolerance to shift in temp depends how fast these changes in temp happens metabolic functions are less sensitive to long term changes in temp than to short term changes acute upper lethal thermal imit exposure to high temp death is relatively rapid chronic or incipient upper lethal thermal imit exposure of organisms to a high temp for a longer period of time Cannot survive sustained exposure Exposure to petroleum affected metabolism and cardiac function of sole Petroleum influence metabolic rate of fish Nicotine tobacco caffeine in terms of tablet light and dark heat cold and solidarity Need to set up proper controls How to set up best control for goldfish Do you use 2 or 4 fish for performing an experiment Run control and experiment with the SAME FISH Do you perform control first and then the experiment or the experiment first then the control The control should be run first and then the condition How to setup repeat experiments Need an N larger than 1 for successful t test Use different set of fish for the repeat experiment This will give you an N of 2 for t test Going to use two fish but fish have different sizes The different fish sizes will produce different data Need to safely handle fish without endangering them Rinse hands with water only before going to lab Book Notes All living things carry out metabolic processes Metabolism total chemical activity within that organism It is the sum of all anabolic and catabolic processes 0 Anabolic pathways synthesize important chemical building blocks Catabolic break down molecules Energy required is obtained through respiration Through respiration organisms obtain oxygen whose rate can be measured to correlate with metabolic rate This can be influenced by an organism39s body temperature Goldfish are poikilothermic organisms body temperature changes when the environmental temperature changes Will used a paired t test to analyze the data Used a small oxygen chamber and because it39s a closed system the chamber will contain less and less oxygen as the fish utilize the oxygen that was initially in the water Variables salinity temperature lighting caffeine nicotine TA Notes Energy required in metabolism is obtained through respiration Respiration requires oxygen Metabolic rate 02 consumedtime Maintaining higher body temp requires higher metabolic rate Homeotherm animal whose body temp maintains relatively constant hard to manipulate ex Dog Poikiotherm animal whose body temp more or less follows ambient relating to the surrounding temperature easier to manipulate eg Frog or fish Lecture 5 PCR analyze DNA to determine your mitochondrion DNA haplotype from your maternal lineage O O O OOOO 3 part series 0 isolate DNA and prepare for PCR 0 samples will then be amplified and sent out for sequencing good primer design is vital part of developing PCR protocol skills micropipeter extraction of DNA from check cells and using BLAST program we will design a primer pair by hand using an online program called primer3 used at crime scenespaternity tests made many technologies possible first conceptualized in 197039s PCR amplification of nucleic acids start with small quantity of DNARNA and then build it up useful to detect mutations in small samples or to detect HIV in blood sample of patient need 1 template amp relatively short sequence 2 pair of primers specifically synthesized 3 enzyme DNA polymerase 4 nucleotides dATP dGTP d39l39l39P dCTP first step to denature at 94 degrees Celsius solution use DNA polymerase from bacterium thermos aquatics called Taq polymerase after DNA melts temperature drops to 50 or 60 degrees which allows primers to anneal only the part between primers is amplified elongation phase 72 degrees Celsius poymerase will extend primer into full length strand same happens on the other side temp9 94 degrees Celsius and Dna melted again separate primer from the template and REPEAT amount of target DNA doubled at each cycle 30 cyces 2 30 target DNA will be dominant sequence 3 most important applications of PCR genotyping diagnostics gene cloning amplified DNA then separate by size using electrophoresis longer DNA fragments run shorter distance on gel top band represents disease allele O O OOOO HIV is retrovirus so core genetic material is RNA ex RT PCR converted to DNA because it is more stable PCR can also be used to access specific sequences to fragment of DNA regions of high variability polymorphic regions which vary in sequence andlength we will use markers on our DNA contained in mitochondria mtDNA circular only from mother one copy only most DNA comes from nuclear genome mtDNA is a haplotype by sequencing your mtDNA you can find out with haplotype branch you belong to hypothesize your haplogroups hyper variable regions is where there are differences for successful primer length is important 2030 bases melting temperature 5580 degrees Celsius G C should make up 5060 of total bp helps determine TM primers should end 339 in G C CG or GC bind more tightly so will increase primer specificity long runs four of same base should be avoided mismatch 9 change in TM but can still extend in 3 mismatch in 3 would make PCR unsuccessful primers work in tandem so TM should be similar problems can fold back on themselves bind to another primer with same sequence and bind to reverse primer Book Notes DNA isolation and Amplification O O humans inherit one complete set of chromosomes from each parent half from mother and half from father Mitochondria also contain DNA small circular molecules that are remnants of endocytosed bacteria Unlike nuclear chromosomes mitochondrial DNA is inherited strictly from the mother Changes in the DNA sequence occur at a low frequency through either replication errors exposure to mutagens or oxidizing conditions as is common in mitochondria Your haplotype is the nucleotide sequence of your mitochondrial genome O O 0 Your haplogroup consists of all individuals with the same haplotype as you You will isolate mitochondrial DNA from your cheek cells and amplify a small region for DNA sequence analysis using PCR This region chosen is a segment within the Control Region specifically the hypervariable segment I located near the origins of DNA replication Since this is a control region not a protein coding region this region of the mitochondrial DNA can accumulate more mutations without affecting function DNA is easily isolated from cells by disrupting the cell wall will do this with heat DNase will degrade DNA These enzymes require Mg2 as a cofactor and we will use Chelex to tightly bind all the Mg2 in the extract and prevent DNA degradation PCR in vitro DNA synthesis reaction repeated many times and uses 2 premade oligonucleotides to amplify a specific region these are called primers The primers are singe stranded DNAs 2035 bases long Primers that are too short are not very specific but primers that are too long will have a harder time binding to the DNA G and C make up 5060 of the total base pairs Primers should end 3 in a G or C CG or GC because GC bind more tightly than A or T Long runs 4 or more of the same base in a row can result in mispriming and should be avoided After about 35 cycles one billion copies of the region between the primers are synthesized Forward primer left primer is identical to the sequence of the reference strand and binds on the complement strand The reverse primer right primer is identical to the complement strand and binds on the reference strand 59 3 reverse complement 3 9 5 complement During PCR the primers will extend from 3 end PCR product size 549 bp TA Notes Mitochondrial DNA mtDNA inherited ONLY from the mother Your specific mitochondrial sequence is called your haplotype Different haplotypes exist due to occasional genetic mutations that occur and are passed down to subsequent generations All haplotypes trace back to a single haplotype called the mitochondrial Eve This is the hypothetical human female from which mitochondrial DNA suggests that all living human evolved Haplogroups are clusters of haplotypes defined by significant sequence changes PCR in vitro always 59 3 starting at a 3 end After everything it can be visualized on an agarose gel and then sequenced The mitochondrial control region contains one origin of replication and 2 promoters It accumulates a high number of mutations since it does not encode proteins DNA is double stranded but we refer to the top reference strand DNA read from 5 9 3 Forward primer is already is 5 9 3 Reverse is 3 95 Lecture 6 Protein and DNA Gel Electrophoresis basic way to understand protein is to know its mass Protein Lab 0 Estimate protein mass 0 Estimate number of subunits of protein mtDNA Lab 0 verify your PCR was success by agarose gel 0 determine DNA concentration by spectrophotometry proteins carry out cell39s vital functions proteins are all made of strings of amino acids proteins polymers of amino acids protein sequences are read from n termina end to the c termina end cysteine is important for determining protein structure all proteins are made of combinations of the 20 amino acids primary structure of a protein specific AA sequence a protein will tend to fold so that its polar and charged residues are exposed to aqueous environment of the cell and interior is made of the hydrophobic environment Most important element of a tertiary structure is a disulfide bond Disulfide bonds can form between 2 cysteines Quaternary final level of protein structures During protein purification we single out one strand of protein by ultracentrifugation separate by size or column chromatography separate by size hydrophobicity charge or binding to specific groups Gel electrophoresis measures the size of a protein Smaller molecules move quicker Coat proteins wSDS which removes weak hydrophobic interactions 0 Interrupts hydrophobic ionic and hydrogen bonds SDS doesn39t disrupt cysteine bridges but bet mercapoethano does Buffer systems enhance the sharpness in a gel DNA doesn39t coil into complex structures like protein We use agarose gel to determine if PCR was successful In Lab will use NuBu instead of Coomassie Blue 9 proteins will use gelred instead of Ethidium bromide 9 DNA TBE buffer Gels and buffer contain material which can cause respiratory tract skin and eye irritation Book Notes Agarose and Polyacrylamide Gels 0 Load sample in polyacrylamide gel and use SDS PAGE technique to determine the size and number of the individual subunits of this protein Some proteins contain more than one polypeptide chain arranged in a quaternary structure Forces that stabilize interactions contain closely packed nonpolar side chains hydrogen bonds and in some cases intermolecular disulfide bonds To estimate the size or proteins can use either gel filtration or SDS polyacrylamide gel electrophoresis SDS PAGE yield different but complementary data Gel filtration estimate of molecular weight of a protein in its native state intact and functional form In contrast the detergent SDS denatures proteins disrupting noncovalent linkages between peptides When SDS is used breaks disulfide linkages that are separated on the basis of differences in their molecular weight using polyacrylamide gel electrophoresis o Electrophoresis is a method for separating charged molecules such as proteins and nucleic acids in an electrical field 0 Rate of a moecue s migration through the electrical field depends on the net charge the size and shape of the molecules 0 SDS page is the most common type of electrophoresis applied to proteins and is referred to as denaturing electrophoresis 0 One molecule of SDS for every 2 amino acid residues 0 Proteins run to the positive ends The smallest and fastest migrating proteins reach the bottom of the gel TA Notes 0 Gel filtration Estimate protein mass in its native conformation 0 SDS PAGE disrupts non covaent interactions Provides similar charge to mass ratio for each polypeptide On a gel proteins of unknown MW are compared to the standard marker SDS PAGE protein gel Agarose DNA gel SDS used to denature protein and DNA is already linear and negatively create uniformly negative charge in charged thus no need to treat a polypeptide further Coomassie alternative NuBu GeRed intercalates between DNA Express base pairs Dye binds to proteins through GeRed fluoresences under UV light basic amino acids Protein binding causes the dye to change from reddish brown to bright blue Vertical Horizontal Higher resolution due to smaller Agarose has lower resolution but pores good enough for purpose of this lab Used to separate molecules of smaller size differences Is a neurotoxin Agarose is safe to handle Gel casting requires air tight Gel casting is easier conditions Both DNA and protein Can run gel Both DNA and protein Can run gel either horizontally or vertically either horizontally or vertically Lecture 7 The Pigments of Photosynthesis Pigments allow organisms to trap light energy and convert it to chemical energy Use thin layer chromatography and spectrophotometry to examine pigment composition of organism and relate results to environmentaIevolutionary factors that influence pigment composition Skills in this lab Thin layer chromatography spectrophotometer wavelengths correctly spotting and calculating Rf values Photosynthesis is process used by plants and other organisms to convert light energy captured from the sun into chemical energy that can be used to fuel organism39s activities Occurs in plants algae many species of bacteria Called photoautotrophs because can create their own food Use Co2 H20 release 02 waste CO2 enters and O2 exits the leaves through openings on the leaf surface called stomata Sugars product of photosynthesis are stored through plant39s body Photosynthesis occurs in organelles called chloroplasts stroma and thylakoid Darker stripes 9 thyakoid lighter 9 stroma Photosynthesis consists of many reactions but are divided in 2 pathways photophosphorylation light reaction and Calvin cycle dark reaction Photophosphorylation is driven by light energy it uses light water and NADP to produce 02 ATP and reduced e carrier NADPH Calvin cycle uses ATP and NADPH made by light reactions and CO2 from environment to produce sugars and other food molecules ATP synthesis and NADP reduction require light so both pathways stop working in the dark Light is a form of electromagnetic radiation and has dual nature Light comes in discrete packets called photons and behaves as if it were a wave Shorter wavelengths are more energetic and longer wavelengths are less energetic Colors 9 visible light Bees can see ultraviolet and snakes can see infrared Molecules that interact with photons in visible spectrum are called pigments Photons must 1 be absorbed by receptive molecule and 2 must have sufficient energy to perform chemical work required in order to interact in biological process When photon meets molecule can undergo o Scattering 0 Absorption causes change to molecule 0 Transmission When a photon is absorbed by a molecule that molecule acquires energy of that photon thereby raised from ground state to higher excited state If excited e does not pass energy we observe fluorescence emission of light by substance that has absorbed a photon Some absorbed energy dissipated as heat so when it goes back to ground state it39s lower in energy again Chloro a does not absorb light in 450550 nm Several diff pigments with different absorption spectra absorb energy that is used for photosynthesis Chlorophylls Carotenoids Phycobilins 0 Last two are accessory pigments Chlorophyll and green pigment found in cyanobacteria and chloroplasts of algae and plants Why are leaves green Reason why most plants look green is because of their red peak and blue peak absorption At center of ring is a magnesium ion and long hydrocarbon tail secures chlorophyll to thylakoid membrane Carotenoids 9 organic pigments found in chloroplasts of plants and other organisms Absorb blue light and have deep yellow color 0 Xanthrophylis Carotenes make carrots look orange precursor for vitamin A They absorb light energy for use in photosynthesis and protect chlorophyll from photo damage Responsible for red yeow colors of leaves in fall green fades away when summer ends Phycobilins 9 found in cyanobacteria and red algae efficient at absorbing red orange yellow and green light which isn39t well absorbed by Chloro a Organism growing in shallow water tend to contain phycobilins that can capture yellowred light and those in deeper water contain phycobilins that can capture green light Used as chemical tags in research by binding phycobiliproteins to antibodies 9 immunofluorescence Photosystems are complexes of pigments and integral membrane proteins Labs green plant light grown barley appears dark in color analyze with TLC move up plate via capillary action endosymbiotic theory proposes that certain organelles are descendants of prok engulfed but not digested by ancient euk Cells double membranes such as those surrounding chloroplasts and mitochondria may have originated when one cell engulfed another all chloroplasts trace their ancestry back to engulfment of one large cyanobacterium by a larger euk cell each major lineage originates from a unique event of endosymbiosis the establishment of stable association between host and either a cyanobacterium or an algae that already harbors chloroplast Book notes o will use two analytical tools TLC and spectrophotometry to test for presence or absence of a variety of photosynthetic pigments Metabolic process where solar energy is trapped and converted to chemical energy and stored in bonds of organic molecules Net result of photosynthesis is that carbon becomes fixed Oxygen gas released as a by product Partitioned into 2 groups Light dependent light energy absorbed and used to produce ATP Light independent energy stored as ATP and NADPH is used to fix carbon dioxide and produce sugars such as glucose o Molecules that absorb light energy in the ight dependent reactions are called pigments o Pigments fall into three classes Chlorophylls Carotenoids Phycobilins o All 3 types can absorb light energy but only chlorophyll can convert this energy directly to chemical energy 0 Carotenoids and phycobilins are known as accessory pigments o Chlorophylls absorb red and blue light to give plants their green color 0 Carotenoids absorb violet and blue light which gives them a yellow orange andor red color 0 Plants only synthesize chlorophylls in the presence of light 0 Phycobilins consist of phycoerythrin and phycocyanin red algae and cyanobacteria o Visible light 400 and 700 nanometers o TLC is technique for separating organic compounds 0 Different components in mixture move up plate at different rates based on size polarity and solubility Pigments absorbed strongly move slowly whereas those absorbed weakly move faster 0 The ratio of the distance moved by a pigment to the distance moved by the solvent is called an Rf TA Notes 0 Chlorophylls Chlorophyll a amp b o Accessory Pigments Carotenoids B carotene and xanthophylls Phycobilins phycocyanin amp phycoerythrin 0 Absorption spectrum is the plot of a pigment s absorption of light versus wavelength of light 0 Each pigment has its own characteristic absorption spectrum 0 Action spectrum plot of biological activity eg photosynthesis versus the wavelength of light OOOOO Chlorophyll is a plant pigment that absorbs light of the blue and red ends of the spectrum Factors affecting pigment composition in a plant Environment 0 Presence or absence light Evolutionary history 0 Closely related organisms share the same suite of photosynthetic pigments TLC used to separate organic compounds Stationary phase often polar 0 Plastic with silica gel Mobile phase often nonpolar 0 Organic solvent mixture Rf is determined by Solubility of molecules to solvent Affinity of molecules to stationary phase Size of molecule Rf value is a unique combination of its Organic compound Mobile phase Stationary phase Rf value is reproducible only when all three variables are the same Light grown very green leaf Darkgrown deep yellow leaf Rat Dissection Proper dissection techniques will be used in this lab Will learn about the various organ systems in detail Rodents are used to study diseases that can39t be studied in humans Knockout mice have been instrumental in developing disease models Specific gene is genetically removed to discover what goes wrong if the gene is missing Metabolic super mouse expresses 100 times normal level of an enzyme involved in glucose synthesis pathway Lab Follow dissection directions from lab manual and computer tutorial 0 Review the directional terms in lab manual to correctly point to organs Dorsal towards backbone The spine is dorsal in the rat Ventral towards the belly of the rat Eg the teats are ventral Posterior towards the back Anterior towards the front Right right of the rat not your right In humans dorsal is equivalent to posterior Deep red color or the liver and pale color of the stomach Mice have 2 carotid arteries lung have 5 lobes 1 and 4 uterus has 2 horns liver has four lobes Humans have One right carotid artery Lung has 5 lobes 2 and 3 Uterus is one muscle Liver has four lobes Book Notes 0 O In humans the anterior coincides with the ventral side of the body Correct path of the digestive system stomach 9 pyloric valve 9 duodenum 9 small intestine 9 ileocolic valve 9 large intestine 9 rectum Overall the length of the small intestine is about six times the length of the body of the rat Look at function of rat structures and functions pg 123 TA Notes 0 Distinguishing features of mammals hair mammary glands with external openings Lips surround mouth philtrum is deep cleft in upper lip Pinna around ears fold to conduct sound Vibrissae whiskers for maintain balance 0 Male rats Scrotum contains testes in ventral posterior part Penis contains opening to urinary and genital organs of the male 0 Female rats Urinary and genital openings are separate 0 Urethral opening separate from vaginal orifice 0 Tail with many scales and few hairs Reproductive system 0 Males Testes in scrotum Epididymis on surface of testes Urethra opens to penis Prostrate gland and seminal vesicle 0 Females Ovaries and fallopian tubes Uterus divided into two uteri called horns Respiratory system 0 Heart has 2 atria and 2 ventricles 0 Pulmonary circulation to lungs systemic circulation to body 0 Locate trachea and inflate the lungs Brain dissection 0 Remove brain from spinal cord 0 Olfactory love in anterior Histology Lab dissecting and compound microscopes will relate structure to function of human cells and tissue samples histology the study of the microscopic anatomy of cells and tissues is important method for analyzing tissues in health disease and research every living thing comes from another living thing microscopic structures of tissues and organs are important to their function at a joint the two bones are in close contact to each other 0 joint undergoes relaxationcompression o the top layer of tissue in knee is articular cartilage O OOOOOOO O OOOO O O OOOOOOOOOOO O OOOOOOOO OOOO the tissue is mostly matrix and not as much cells canals in bone contain blood vessel and marrow Masson s trichome stain is common Hematoxylin is known as an acidophilic dye meaning it is attracted to acidic substances such as nuclei acids Stains nuclei dark purple Eosin is also called a counterstain It is a basophile and stains alkaline parts of the cell such as cytoplasm Red orange pink we will use E amp H in lab for staining Cat ovary huge cell with a tiny nucleus Histology is also important in the diagnosis of disease histopathology Most common method is called a biopsy A small sample of tissue is surgically removed from a suspicious site Cancer cells grow rapidly are very metabolically active Pap smear done to make sure no cells show evidence of cervical cancer Coonoscopy look in rectum to check for colon cancer Histology can be used in research as well Teratoma assay suspected stem cell inject in mouse to see if teratoma forms Teratoma type of tumor that contains many diff types of tissues Can be used to find out if the stem cell is pluripotent able to differentiate into tissues from all 3 germ layers Ectoderm Mesoderm endoderm Immunohistochemistry antibodies used to mark specific proteins in the tissue slides Lab using microscope to examine series of slides deduce which tissue each slide corresponds to predict what the cells will look like from a box of 9 slides electron microscope magnification power of 2 million x no living samples using beams of electrons to obtain image light microscopes compound microscope dissecting microscope compound microscope two lenses focus on light that passes through object requires thin specimen produces mirror image discrete increments of magnification powers 1000x dissecting microscope focuses on light reflected off the specimen image has same orientation magnification 50x continuous range of magnification 0 will be operating these 2 microscopes o examine the macro samples provided 0 follow the computer tutorial 0 identify the 9 unknown slides but identify eight tissue types 0 liver 0 lung 0 trachea 0 muscle 0 thymus 0 brain 0 blood 0 bone 0 you will see purple and pink structures 0 RNADNA will be stained dark violet o Cytopasm slightly pink 0 Histology slides made of glass and they can break if you break a slide notify the TA so she can take it to the prep room 0 Dispose of glass in glass waste container TA notes Blood red blood cells Cluster of cells that contain no nuclei Hundreds of round disks with no nucleus purpe coored disk ike cells Bone Cells that were porous in places with nuclei that were spread out Pocketsholes for blood vessels or nerves Liver Similar structure to thymus with less nuclei Soft amp smooth highly backed with blood cells Lung Has a lot of air bubbles mostly pink some nuclei surrounding air pocket Round microscopic pouches known as alveoli Neurons Can see the cell body axon that are connecting different neurons Skeletal muscle Striations within the tissue sample and parallel fibers Skin Layers of different types of cells Starts thick and gradually gets thinner in cell density Thymus Cells very densely packed with nuclei 0 Hematoxylin basic alkaline dye Is positively charged Bluepurple in color 0 Eosin acidic dye Negatively charged Pinkred in color Electron microscopes 0 Using beams of electrons to obtain image 0 No living samples Light microscopes 0 Compound microscope 2enses focuses on light that passes through the object thin specimen mirror image discrete increments of magnification powers 40x 100x 400x etc o Dissecting microscope Focuses on light reflected off the specimen Image has same orientation Magnification 50x Continuous range of magnification Used for gross morphological examination is used mostly on larger specimens Works like a zoom lens of a camera Book notes o Histoogy study of tissues o Cell structure is tied to function 0 Lecture 10 DNA Sequencing Analysis final part of investigation into DNA history Squot3939gtSquotquot will clean up raw data in trace editor estimate haplogroup validate haplogroups annotate mtDNA sequence align with similar sequences create a phylogenetic tree with your sequence there are some current existing models of divergences of Human Gene when modern human left Africa there were hominids living in many different places haplotype specific nucleotide sequence of mitochondrial genome haplogroup all individuals with the same haplotype along with other haplotypes that are very similar DNA Sanger sequencing requires a number of reagents O O 0 Template Polymerase Sequencing primer will be extended by pol that can sometimes be radioactively labeled Nucleotides used to build the DNA ddNTPs N stands for the four bases Lack a 3 OH group Since new nucleotides are added to the 3 OH when a ddNTP is introduced it causes DNA synthesis to stop ex in addition to 4 standard nucleotides there is small amount of ddGTP whenever this is incorporated it will cause DNA synthesis to terminate Next need to run this mixture on a DNA gel Will use a polyacrylamide gel to give a very high resolution The products closer to bottom of gel are shortest and fastest moving fragments Automated sequencing Now most samples can be processed in a short amount of time Can label the ddNTPs with colors to distinguish which base each fragment will end with and can be deciphered with a computer the process will be automated To sequence a whole genome other technologies are available Most successful technology is Illumina or Solexa New technologies use non iving substrates Genome sequencing 0 Understand the basis of diversity 0 Understand evolution BLAST compares sequences Mega5 will take multiple sequences and find the best alignment and then trees can be built Receive the DNA sequence data as a four color data one different color for each kind of nucleotide Peaks are sharp and strong at beginning of the run Towards the end the peaks get broader and weaker We are expecting 529 base pairs from the sequencing run Will use BLAST alignment as a guide Top seq is query sequence and the bottom is the reference seq The alignment will automatically drop bases that don39t match Empty spaces in between bases indicate differences To annotate means to put a note on a piece of DNA about it39s content Book Notes DNA sequencing like PCR is based on in vitro DNA synthesis Sanger sequencing synthesis of new DNA terminated by addition of a ddNTP because they have a hydrogen in the 3 position instead of a hydroxyl group and thus stop DNA synthesis when added to a growing chain The data printout is called electropheroqram or chromatoqram 4514 934 PM
Are you sure you want to buy this material for
You're already Subscribed!
Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'