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103 Cards in this Set

  • Front
  • Back
What is molecular biology?
the study of DNA and proteins.The study of molecular information transfer within the cell, and understanding pathways. It overlaps with biochemistry and genetics.Includes the analysis and manipulation of DNA and proteins using many techniques.
What are PPE?
(Personal Protection Equipment) A barrier method of protection in the lab. This includes gloves, UV eye protection, lab coats, pants, etc.
What is an MSDS?
A Material Safety Data Sheet. It states a chemicals physical and chemical properties and any associated physical hazards, health hazards, the primary route of entry, exposure limits, and whether or not it is toxic, carcinogenic, flammable, or radioactive. It also lists proper safe handling of the chemical, and emergency and first aid procedures.
What are the OSHA requirements?
~ employers must inform employees of potential hazards.
~ requires that every hazardous substance has a Material Safety Data Sheet
What can you use a p10 Denville micropipette for?
To dispense amounts from 0.5ul to 10.0ul
What can you use a p20 Denville micropipette for?
To dispense amounts from 2.0ul to 20ul.
What can you use a p100 Denville micropipette for?
To dispense amounts from 10ul to 100ul.
What can you use a p200 Denville micropipette for?
To dispense amounts from 20ul to 200ul.
What can you use a p1000 Denville micropipette for?
To dispense amounts from 100ul to 1000ul.
What can you use a p10 Eppendorf pipette for?
To dispense .5ul to 10ul
What can you use a p100 Eppendorf pipette for?
To dispense amounts from 10ul to 100ul.
What can you use a p1000 Eppendorf pipette for?
To dispense 100ul to 1000ul.
What are preparative centrifuges used for?
They are used to separate molecules and can help to isolate or purify a sample
What are analytical centrifuges used for?
They are used to analyze the sedimentation characteristics of pure biological macromolecules and molecular structures.
What is a large capacity centrifuge used for?
for larger volumes, up to 1 liter per bottle, and can reach around 600 rpm and 6500 g. These are used for separation of large amounts of material, like liters of bacteria from media.
What is a high speed centrifuge used for?
used for large volumes needing even higher speeds, and can reach about 25,000 rpm and 60,000 g for the collection of microorganisms, cellular debris, organelles, or proteins precipitated by ammonium sulfate. Sample volumes are usually smaller (50 ml tubes).
What are preparative centrifuges used for?
They are used to separate molecules and can help to isolate or purify a sample
What are analytical centrifuges used for?
They are used to analyze the sedimentation characteristics of pure biological macromolecules and molecular structures.
What is a large capacity centrifuge used for?
for larger volumes, up to 1 liter per bottle, and can reach around 600 rpm and 6500 g. These are used for separation of large amounts of material, like liters of bacteria from media.
What is a high speed centrifuge used for?
used for large volumes needing even higher speeds, and can reach about 25,000 rpm and 60,000 g for the collection of microorganisms, cellular debris, organelles, or proteins precipitated by ammonium sulfate. Sample volumes are usually smaller (50 ml tubes).
Describe the types of spectroscopy.
infrared, fluorescence, nmr, x-ray, flame, cd
What is Spectrophotometry ?
involves measuring a certain wavelength using prisms or gratings and measures the spectral properties of molecules.
What is absorbance?
absorbance is the measurement of how much light is absorbed by the mixture.The larger an absorbance is the more concentrated a solution is, and vice versa.
What is transmittance?
Transmittance is the measurement of how much light passes through a mixture Out of 100% absorbance + transmittance = 100%
What is the importance of an absorbance value measured at 260 nm?
This is the wavelength of light that is used to measure the DNA concentration/absorbance.
What is the importance of an absorbance measured at 280nm?
This is the wavelength of light used to measure the absorbance/concentration of protein in a solution.
What is Beer''s law?
Mathematical formula that defines the relationship between absorbance and transmittance. A = bc = log (I0/I) = log (1/T) = -log T
What is Protein quantification ?
a method for determining the amount of protein in a given sample.
Describe amino acid analysis.
The most accurate technique of protein quantification.protein is analyzed amino acid by amino acid via hydrolysis, and the protein’s identity and concentration are determined
What are calorimetric methods of protein quantification?
These are methods that involve measuring color changes.
Describe the Bradford Assay.
A colormetric method that involves using Coomassie blue. It changes from red 465 to blue 595. It does not detect free amino acids however, but it forms string noncovalent bonds with acids. Does not work well with proteins below 3000kDa.
Describe the Lowry assay.
Uses copper binding to peptide bonds, a folin reagent is then added and the solution then turns a blue color, then the absorbance/transmittance value is measured at 750nm.
What is the DC protein assay?
It is the detergent compatible version of the Lowry method.
What is the purpose of a standard curve during protein quantification?
A standard curve is used to help determine the concentration of the sample after obtaining a measurement of the sample's absorbance. This is used during protein quantification.
What is Molarity?
can be calculated by dividing the amount of solute (in moles) by the volume (in liters).It is the measurement of the strength of a solution.
What is percent composition?
They are used as an alternative to molarity. They are often w/w, v/v, or w/v.
Describe the enzyme-substrate model.
The enzyme is a protein that is able to lower the activation for a reaction to proceed. The substrate is something that binds to the active site in the enzyme and allows for a conformation change and allows for formation of a product.
What is a lab protocol?
a list of steps that should be followed to obtain the desired results, should include everything you need to reproduce the experiment exactly and be as specific as possible
What is a peer review?
A peer review is when a paper is submitted to be reviewed by scientists in the field to be criticized and analyzed. Then if two-thirds of them come back favorable the paper is usually published.
What is a double blind experiment?
the researcher does not know which is the control group and which is the experimental group. This technique is often used when a new drug undergoes human trials – the doctor prescribing the drug does not know whether it is the actual drug being tested, or a placebo.
What is Scientific rigor?
the data and conclusions are supported across all tests, and withstand any experiment they are put through.
How is cloning done?
Cloning involves using a vector to create a piece of recombinant DNA.This recombinant DNA is inserted into the cell of an organism and the cell of that organism replicates also replicating the inserted vector.
What is recombinant DNA?
It is a plasmid (usually) that contains inserts from various organisms, and is not found in the environment.
What is the pET vector system?
a controllable expression system that uses host proteins, bacteriophage proteins, and the lac operon.
Describe the IPTG induction system.
the lac repressor is removed from the operator, and E. coli RNA polymerase and ribosomes transcribe/translate T7 RNA polymerase. T7 RNA polymerase finds the T7 promoter on the plasmid, transcribes the gene of interest into mRNA, which is translated to protein by E. coli ribosomes. The end result is high expression of a recombinant protein
What is T7 RNA polymerase?
RNA polymerase that catalyzes the formation of RNA in the 5'→ 3' direction. T7 polymerase is extremely promoter-specific and only transcribes bacteriophage T7 DNA or DNA cloned downstream of a T7 promoter.
Describe protein purification.
It is a technique that is used to isolate a specific protein from a lysate.
Describe the ammonium sulfate cut process.
a controlled precipitation in a sense, in that protein is gently removed from the buffer and stabilized by the positive charges of the ammonium and negative charges of the sulfate.
Describe how dialysis works.
A process in which proteins are placed in a special membranous tubing with very small holes that only allow the buffer through.
Describe Protein concentration.
involves removing some of the buffer from a sample while keeping the protein of interest. In this way proteins can be concentrated from a very low starting amount (g/mL) to 10-100 mg/ml or higher if the protein will tolerate such concentrations.
What is lyophylization?
the process of drying proteins by using a vacuum.
What is ultrafiltration?
proteins are placed onto a membrane that only allows for the buffer to run through it so what is left on top of the membrane is the protein.
What is SEC?
Size exclusion chromatography. It allows for purification of nucleic acids, sugars, and proteins. Sample must be applied to the top of the column, bad broadening may occur.
What flows through in SEC first?
The larger molecules would flow through first.
What flows through SEC last?
The smaller molecules take longer and get slowed down by the ores in the beads, so they will elute last.
What is IEX?
uses a column just like SEC but also allows for the separation of a sample based off of charge.
What is an isoelectric point?
the pH at which there is an overall net neutral charge on the molecule.
Describe protein binding and charge.
A protein will not bind to a charged column of any type if in a buffer with a pH of the protein’s pI. As the pH increases above the pI, protons dissociate from the protein’s termini and side chains, leaving a net negative charge on the molecule. If the pH decreases below the pI, protons associate with the protein, giving it a net positive charge. A protein will bind to a column of opposite charge.
What is an anion exchanger?
those which chemically bind negative charges
What is a cation exchanger?
a column that binds positive charged proteins.
Anion exchange chromatography.
exchanging negatively charged anions, which is accomplished by using beads with a positive charge.
Cation exchange chromatography.
involves exchanging positively charged cations, which is accomplished by using beads with a negative charge.
What is Affinity chromatography?
involves one molecule binding specifically to another molecule in a sample. The beads of an affinity column contain an immobilized molecule that binds with high specificity to a single protein or a small number of similar proteins, nucleic acids, or other molecules.
What is the gene fusion technique?
the gene encoding the protein of interest is fused to a second gene encoding a protein that is easily purified by affinity chromatography.
What is metal chelate chromatography?
immobilized metal affinity chromatography, or IMAC) in which nickel or cobalt are loaded onto a column and proteins with histidine (his) tags are passed over the column. gene of interest is genetically manipulated to encode a polyhistidine tag, usually six to eight histidines in length, at either the N- or C-terminus of the protein.
What is SDS-PAGE?
~Sodium dodecyl sulfate polyacrylamide gel electrophoresis
~ was developed to allow proteins to separate by only a single factor – molecular weight. It is used for the routine estimation of protein identification, purity, and quantity. It can be used to track the course of purification of proteins for which there is no convenient enzymatic assay, and allows one to visualize and track both the protein of interest and contaminants. SDS-PAGE can resolve between two proteins that are approximately 1% apart in mass.
How is Coomassie Brilliant Blue used?
~ Used to visualize bands in the gel.
~ soaked in coomassie and destained with methanol.
Silver Staining.
~10-100X more sensitive than coomassie
~ Silver atoms bind tightly to proteins and produce black or purple bands.
Describe autoradiography.
~ The most sensitive type of gel detection
~ involves the uses of radioisotopes and X-ray film
What is native gel electrophoresis?
researchers to run proteins through a gel without denaturing them, therefore retaining their native structure and activity.
Which properties are the proteins separated by in native gel electrophoresis?
~proteins will carry a charge relative to their pI and the pH of the buffer. Because of this, proteins will migrate relative to both their charge and size.
~The buffer used is usually around pH 9, so most proteins will carry a negative charge and migrate towards the positively charged electrode. But this is not always the case, and proteins with very high pIs will migrate in the reverse direction, towards the negative electrode.
How can native gel electrophoresis be useful and what can be determined from it?
Native gel electrophoresis allows one to determine a protein’s conformation, quaternary structure (oligomeric state), analyze its structure/function, study protein-protein interactions, perform enzyme assays directly on the gel, and study protein modifications (phosphorylation, glycosylation, activation).
Describe isoelectric focusing.
allows the separation of proteins on charge alone. At a protein’s isoelectric point (pH = pI) it will not move in an electric field. This property is exploited by isoelectric focusing, which separates proteins by their pI values.
Two-dimensional (2D) gel electrophoresis.
a combination of IEF and SDS-PAGE. A tube IEF gel is run, and the resulting tube gel is then placed horizontally across the top of the stacking gel of a slab SDS-PAGE gel. The proteins are separated by their pIs in the first (IEF) dimension and according to their molecular mass in the second (SDS-PAGE) dimension.
Describe the ELISA.
~ enzyme-linked immunosorbent assay
~allows for the detection of an antigen or antibody in a sample. It is often used as a quick medical diagnostic tool to detect the presence of certain protein markers or viruses in a patient, in industry to check sample quality, and in pregnancy tests.
~ nearly identical to a western blot except that an SDS-PAGE gel is not run. Instead, samples are placed in small wells and assayed, often many at a time. If a reaction occurs, the well will change colors, indicating that an antibody is binding to an antigen. While very sensitive, this technique is at a disadvantage over the western blot because it provides no information on the molecular weight of the antigen. Therefore, an antibody in an ELISA could bind nonspecifically and it would be difficult to detect.
Immunoprecipitation.
involves precipitating an antigen out of solution using an antibody as a purification step
~the antibody will bind to the protein of interest and it can be spun and washed. The protein can then be run on SDS-PAGE, mass spectrometry, or a western blot for identification.
co-immunoprecipitation
~ aka: pull down
~involves binding an antibody to a protein on solution, hoping to pull-down other proteins in complex with it to detect new protein-protein interactions.
~the protein bound to the antibody will hopefully interact with its binding partners in the cell extract, and you can pull them out of solution together (hence the name “pull-down”) and analyze the sample.
Protease digestion
technique that allows for protein analysis, including domain analysis, conformation and activation analysis, and protein fingerprinting. A protease is an enzyme that cleaves a protein at a specific amino acid sequence
mass spectrometry sequencing
a protein is digested, and the molecular weights of are analyzed and compared to known fragments. The protein’s sequence can be determined by overlapping the fragments.
far-western blot
allows one to identify protein-protein interactions. It is essentially a western blot with a few minor differences, such as a native gel (as opposed to SDS-PAGE), and a radiolabeled bait protein probe instead of an antibody. If the bait interacts with any proteins on the gel it will bind and can be detected with autoradiography.
yeast two hybrid system
a large library of potential partners can be screened. In the event of protein-protein interactions, the system uses a reporter gene (lacZ) that is transcriptionally activated and produces a color change in the yeast. As shown in the figure, a bait protein (X) is fused to the binding domain (BD) of Gal4, a transcriptional activator that binds DNA and activates RNA polymerase. A library of binding partners can be screened, and if a partner (Y) interacts with X, the activating domain (AD) will interact with RNA polymerase, triggering reporter transcription.
calorimetry
allows for the measurement of heat change when two proteins bind one another. There are two types. Isothermal titration calorimetry measures heat change upon binding when one protein is titrated into a tube containing a binding partner (such as a peptide and a protein that interact). The second, differential scanning calorimetry, changes the temperature and measures the results, such as when comparing a protein vs. a mutant. The data from calorimetry allows for the calculation of free energy (G), entropy (S), enthalpy (H), molar ratios, and dissociation constants (Kd or “tightness” of binding) of binding partners.
surface plasmon resonance
involves immobilizing protein on a membrane, running the binding partner across it, and after a certain amount of time add buffer with no binding partner across it and measure the rate of what associates vs. what dissociates to determine the Kd. Or in other words, saturate the system, and then measure the time it takes for the molecules to dissociate.
Describe the Southwestern.
allows one to search for proteins that bind DNA by using SDS-PAGE of the protein, removal of the SDS, refolding of the protein with urea, and blotting it onto nitrocellulose. After this, digested labeled DNA is added, and DNA-binding proteins can be identified. This technique is very useful for identifying tissue-specific DNA-binding proteins.
Describe the EMSA.
~electrophoretic mobility shift assay
~involves running DNA or RNA on a gel, followed by DNA with a potential binding partner and looking for a shift on the gel. If observed it indicates a protein-DNA interaction. The protein can be identified by using an antibody and looking for further shifting.
miniprep
~ One of the most common DNA purification techniques
~ based on the alkaline lysis technique of DNA purification, but also involves a silica-based column that will selectively bind DNA. The goal of a miniprep is to lyse bacteria like E. coli and remove everything (including genomic DNA) while collecting plasmid DNA. The end result is very pure plasmid, which can be stored for years in a freezer for preservation, or used immediately for cloning or PCR.
Describe RNA purification.
involves guanidinium isothiocyanate which denatures proteins and inactivates intracellular RNases. Following this a phenol-chloroform extraction and centrifugation are performed to purify RNA from DNA.
agarose gel electrophoresis
allows for protein quantification, a technique is needed to quantify your sample of DNA. Overall an agarose gel is very similar to SDS-PAGE (add sample buffer, load sample in wells, run the sample towards the positive red electrode)
Describe migration in agarose gel.
Smaller fragments migrate more quickly while larger fragments take longer. Since DNA is negatively charged it will run towards the positively charged electrode.
What are probes?
Probes are 32P- or fluorescence-labeled ssDNA that can hybridize (base pair) to a piece of ssDNA in a sample and tell you if that sequence is present or absent.
What is a dot blot?
~The most basic application of a probe to determine whether a given sequence is present or not in a sample of DNA.
~DNA is pipetted onto a membrane, denatured to single-stranded DNA via NaOH and heating, and a labeled probe is added. If the probe binds to the sample it can be detected by autoradiography by the presence of a dot.
What is FISH?
~fluorescence in situ hybridization
~Metaphase chromosomes denatured and mixed with a labeled probe that will bind to a specific sequence of DNA. If this DNA is present its position on the chromosome can be identified with a high degree of accuracy – within several thousand base pairs of the exact location.
What is a southern blot?
~ a technique that not only tells you whether a piece of DNA is present or absent, but it also tells you the size of the DNA
What is RFLP?
~ Restriction fragment length polymorphism
~ an application of the Southern blot that allows one to identify whether an individual has a certain gene, or a different allele or mutant of this gene. The technique is based off of the fact that there are polymorphisms in populations – single nucleotide changes at various locations in the genome.
What is VNTR analysis?
~ Variable Number of Tandem Repeats
~ relies on the fact that DNA polymerase tends to slip in regions heavy in repeats (i.e. ATATAT, or CGACGACGA) when copying DNA. It is essentially undetectable to DNA polymerase because as the template and new strand slip they still line up perfectly with one another.
Describe northern blotting.
almost identical to a Southern blot, except instead of searching for DNA you are searching for RNA. A labeled DNA or RNA probe is used. A cell lysate from a tissue or organ could be tested for the presence of a specific mRNA, and the amount of mRNA present would indicate the amount of a specific protein that is being expressed in that cell type.
Describe a DNA microarray.
thousands of probes are covalently linked to a glass or silicon chip. The sample is washed across the chip, and if the DNA is complementary to a probe it will hybridize to that probe. This hybridization can be detected via fluorescence or a laser, and a gene’s presence or absence, or mRNA expression levels can be determined.
What is meant by transformation?
~transfection in eukaryotic cells
~competent cells which can take up DNA from the environment must be used
How is CaCl2 used in transformation?
For a successful transformation to occur the cell must be treated with CaCl2 and electroporated to allow for the cell to take up the vector.
What is phenotypic screening?
used when cloned gene is expressed and changes properties of the cell in an obvious way (i.e. changing its color).
What is antibody screening?
the cells that contain the vector and gene of interest are expressing a protein to which an antibody is available
What is hybridization screening?
useful if some part of the nucleotide sequence of the gene of interest is known. Cells can be transferred to a nylon filter, lysed, treated to produce ssDNA, and a labeled probe can hybridize to the gene of interest.
What is blue/white screening?
White colony has the insert while the blue colony lacks the insert.