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102 Cards in this Set
- Front
- Back
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What does protease do?
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degrade protein
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DNA nuclease
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digests DNA eliminates transormation
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recombinant DNA
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inserted pieces of DNA into a vector
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What is a vector?
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DNA that is easy to work with and has selectable genes
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5 steps to generate recombinant DNA
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1. Isolate DNA - both source and vector
2. Restriction digestion - make smaller pieces of source DNA and creates a cloning site in the vector DNA 3. ligation reactions - insert vairous pieces of source DNA into the vecotr DNA 4. Transormation - uptake and epxression vector DNA into a host, typ eubacteria 5. Screen host cells fro those that have recombinant vector DNA that contains yoru gene of interests |
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What is a common method to isolate DNA
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1. lysing cells with detergents.
2. treated with protease and RNAase 3. lysate treatd with phenol 4. dailyzed to remove low mol weight 5. ehtanol added and DNA precipiates |
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What is fucntion of restriction enzymes?
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fucntion as immune system for prokaryotes.
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what is palindromic?
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each strand reads the same in 5' to 3'
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What is the benefit of using restriction enzymes?
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They cut at specific sites. allows scientists to manipulate and recombine DNA
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What is transormation?
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a way of "getting" the plasmid into the bacterium and having it expressed
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Ligate
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covalently link
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vector DNA
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typically plasmid or viral
cannot replicate itself and must find its way into a host |
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Advantages of vectors
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1. # of base pair is few minimize damage to DNA
2. replicates independently and can be found in large # 3. contain selectable genes |
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What is the most widely used bacterium for vectors?
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Escherichia coli
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What is the most common used virus for vectors?
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lambda virus
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bacteriophage
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virus that infects a bacterium
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lytic cycle
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bacterium lyse after being infected, thus releasing the viral progeny
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What is the prophage stage?
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when the viral DNA is incorporated into its chromosome
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describe plasmids
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double stranded closed circular molecules of DNA found in cytosol
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What are benefits of plasmids?
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anti biotic resistance
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What does pBLU have a resistance for?
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ampicillin
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Ampicillin
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antibiotic which inhibits cell wall synthesis in eubacteria.
Eubacteria containing pBLU produce protein, beta-lactamase that chemically modiefies ampicillin. |
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What restriction enzyme do we use in GMB I?
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HinD III
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What are the 3 classes of plasmid for GMB I?
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1. re-ligated plasmid with no insert
2. two types of plasmids with an insert, either your gene of interest or 3. not your gene of interest |
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Why do we only have 2 classes in our ligation reaction?
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b/c they were purified
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LB
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Luria Bertani Broth
contains primarily glucose as an energy and carbon source |
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What is the purpose of "agar" in LB?
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"gels" the medium such that a solid surface is formed, and bacteeria can be immobilized onto solid medium
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What are the two markers of pBLU?
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ampicillin resistance and beta-galactosidase activity
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What does ampicilin resistnace tell us?
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differentiate btwn transformed and non-transformed
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What does beta-galactosidase activity tell us?
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diffrentiate between plasmid DNA with and without an insert
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What are features that make pBLU an effective cloning plasmid?
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1. origign of replication
2. selectable markers (antibiotic resistance, color selection) 3. multiple cloning site |
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What color is the colony if the host contains ampicillin resistance?
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blue
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How does it turn blue?
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beta-galactosidase, an enzyme conversts galactose anolog, X-gal, into blue pigment
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What is the goal of the GMB lab?
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To insert foreign DNA into the MCS. the insertion destroys beta-gal activity.
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Common characteristics that all organisms share
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1. all organsims made up of single cell or many cells.
2. cells have accurate and programmed mech for repoduction and metabolism 3. cells come from pre-existing cells on a human time scale 4. all cells are ogranized according to fund euk and prok pattern. |
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5 kingdoms
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Monera, Protista, Animalia, Fungi, and Plantae
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three domains
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bacteria, archaea, and Eukarya
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Archaea
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membrane bound. posses nucleus which is membrane-bounded.
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Waht do all organisms do similarily?
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acqure energy and raw materials
transform energy and raw materials reproduction |
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what two domains does prokaryotic organisms fall into?
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BActeria and Archaea
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autotrophic
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photosynthetic
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chemoautotrphic
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oxidize inorganic substance for energy
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mesosomes
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invaginations, increase the surface area of the cell membrane
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3 shapes of prokaryotes
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1. cocci (spherical
2. bacilli (rod shaped) 3. spirilli (spiral shaped |
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gram positive prokaryotes
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the cell wall retains violet color
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gram-negative
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red b/c crystal violet iodine is rinsed away replaced wit hsafranin red.
thin walls tend to be more threatening |
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4 kingdoms that eukaryotes belong to
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Plantae, Animalia, Fungi, and protists (single cell)
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What is the purpose of methocel?
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to slow down organisms
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4 tissue types
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epithelium, connective tissue, muscle tissue, and nervous tissue
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Plants consist of three tissue systems
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1. ground tissue system
2. vascular tissue system 3. dermal tissue system |
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Ground tissue consists of three cell types
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1. paranchyma
2. collenchyma 3. sclerenchyma |
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Vascular consists of what 2 things?
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xylem and phloem
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heterocysts
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specialized cells found in cyanobacteria responsible for fixation of Nitrogen
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amylase
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enzyme hydrolyzes starch
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What are two functions of DNS?
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1. extreme alkalinity denatures amylase
2. indirectly allows you to measure the amount of maltose |
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How does DNS indirectly allows to measure maltose?
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DNS reacts with aldehyde groups. each maltose formed as one aldyhde group that can react.
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What to add for Blank?
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add amylase, then DNS, and finally starch.
DNS denature amylase b4 it can react with starch |
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OD units conversion
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0.4 OD units = 1 micromole of maltose
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affinity equation
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1/Km
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Km value is equal to what?
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equal to the amount of substrate at whcih the enzyme is operating at 1/2 its max velocity (Vmax)
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HOw do you calculate how much activity in undiluted saliva?
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i dont know really
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Why did we use amylase for the lab?
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easy to work iwth and there are several different forms or isoszymes
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Do Enzymes change delta G of reactions?
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NO
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What does enzyme do to the activation energy?
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lowers it.
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When are carbohydrates called monosaccharides? polysaccharides?
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if they are made of only one simple sugar. polymers of many simple sugars
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maltodextran
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maltose and polymers of various sizes
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phosphorylase
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breaks down glycogen
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beta amylases occur only in plants or animals?
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plants
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isozymes
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have variations in both structure and catalytic characteristics
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what does saliva consist of?
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amylase, water, other proteins, mineral salts, and mucin
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mucin
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viscous glycoprotein
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Why are many sugars classified as reducing sugars?
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b/c they contain reactive ketone or aldehyde groups, capable of redicing certain compounds
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Why is it that extremely large polysaccharide such as startch, does not act as reducing sugar?
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only a few "loose ends" less ketone or aldyhde groups.
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enzyme-substrate affinity
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a concept which characterizes the relationsihp between an enzyme and its stubstrate
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low affinity enzyme
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same as low sensitivity
reaction proceeds at a respectable rate only when substrate is present in high concentration |
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hgih affinity enzyme
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high sensitivity
enzyme reacts even if substrate concentration is low |
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what does low Km suggest? high Km
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low Km is high affinity and high Km value is low affinity
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List three major effects temperature has on enzyme catalyzed reaction.
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1. mkinetic largely determines the rate for both forward and reverse rxn. temp is measure of kinetics
2. the addition of heat or removal of heat can result in one rxn rate becoming greater than the other 3. temp affect shape of proteins |
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is denaturation a reaction
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YES!!!!!!!!!!!
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T or F
the duration of exposure to high temp is not equally as significant as the actual temp of exposure. |
FALSE
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List two major affects that pH has on enzyme catalyzed reactions
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1. may cause denaturation
2. may affect reaction rates without causing denaturation |
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where does carbon fixation occur?
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stroma
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photosystem consists of two parts
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antennae and rxn center located thylakod membrane
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proton gradient in cholorplast is from what to what?
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high concentration (lumen) to low concentration (stroma)
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NADPH and H and ATP is used to reduce CO2 to what?
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CH2O by the carbon fixation reactions. Calvin cycle
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How could you measure photosynthesis?
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follow changes in concentration of either substrates or prodcuts. the lab you use DCPIP
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Why do we use DCPIP
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in vivo NADP+. it is blue, but the reduced form, DCPIPH2 is colorless.
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During ETC where are the protons being pumped?
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pumped from the stroma into the lumen
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Where does the Calvin cycle occur?
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stroma
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What is the purpose of using methylamine (CH3NH2)
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acts as pH buffer to minimize changes in proton concentration in the lumen
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what is purpose of having sucrose?
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acts as osmoticum, which prevents the chloroplasts from lysing
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How do you measure the rate of photosynthesis?
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measure rate of photosynthesis by measuring rate of color change of DCPIP
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What are u measuring? the light or "dark" reaction?
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light reaction. (PSI and PSII ETC)
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List three conditions you will measure photosynthetic rates
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1. darkness
2. light and DCMU 3. light 4. light and methylamine |
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What is DCMU?
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ingredient in commerical herbicides and it blocks electron transport between PS II and PS I.
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Why is methylamine used in the test for photosynthetic rates?
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pH buffer minimizes changes in the proton gradient in the thylakod lumen. Without proton gradient cannot make ATP
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Why doesn't thylakoid membranes rupture?
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due to the presence of galactolipids
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What does methylamine do to ETC?
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increases it. effect is known as "uncoupling" of ATP formation form electron flow
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HOw do you make absorption spectrum of purified pigments?
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obtain similar band from several chromatograms and "pool" them together. add actone extract pigments. then measure aborbance every 20 nanometers.
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What do acetone extracted pigments do when irradiated with UV light?
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release energy as heat and fluorescence.
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oxygen liberated in plant photosynthesis comes from what?
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H20 dumbass!
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calvin cycle forms what products?
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sugar and starch
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