UNIT 9 - Techniques Flashcards
Explain the principle behind causing cell lysis by lowering the ionic strength of the media.
Lowering the ionic strength of the media will cause cell lysis, because the water in the media will flow into the cell where the concentration of ions is higher in order to balance out the concentration. The rush of water into the cell by diffusion will cause the cell to burst.
What is lysozyme? What does it do? Why is it used for bacterial cells?
Lysozyme is an enzyme that can break down bacterial cell walls. It is used because bacteria have cell walls that are resistant to lysing by just lowering the ionic strength of the media.
Define the term “centrifugation”
Centrifugation involves spinning of a sample at a high speed, which results in separation of cell components by density.
Define the term “pellet”
A pellet is the part of the sample that is the sediment after centrifugation
Define the term “supernatant.”
the supernatant is the part of the sample that is the liquid at the top of the tube, or the remainder in the tube after the sample is pelleted.
How is centrifugation used to separate cellular components?
Centrifugation is used to separate cellular components because spinning of cells at varying high speeds allows for separation of components by density or mass. Multiple centrifugation steps involving pipetting off the supernatant and re‑centrifugation will allow a differential separation of cellular components to a certain extent.
What is the basis for chromatography? What are the general components? How does it generally work? What is the purpose of this technique?
Chromatography allows the separation of molecules to a higher resolution and may allow purification of one molecule such as a protein. It is performed in columns or tubes containing a material used for separation based on size, charge, polarity, or molecular interactions. A buffer is added, and the sample is then added and allowed to flow through the support matrix. Different tubes or fractions are collected as the sample flows through and one of the fractions should contain the purified molecule.
Say you are interested in purifying a molecule with a known negative charge. What type of ion exchange chromatography would you use? What would be the charge of the counter‑ion you would use?
To purify a molecule with a known negative charge, you would need to use anion exchange chromatography. The charge of the counter ion would be negative.
What is a fraction? Do the molecules of interest come out in earlier or later fractions from an ion exchange column? Why?
A fraction is a sample that is collected in a tube as the sample solution is allowed to flow through the column. The molecule(s) of interest come out in later fractions from an ion exchange column because the molecule of interest would first stick to the column, allowing other components to flow through first and later be released after adding the counter‑ion to the column, which displaces the molecule of interest that is stuck to the column.
What is the support made up of in gel exclusion chromatography? How does this allow separation of different‑sized molecules?
The support in gel exclusion chromatography is made of gel beads with small holes in them that allow only the passage of molecules of certain sizes. The size of the hole is called the “exclusion limit.” Molecules larger than the exclusion limit in a mixture will not enter the tunnels; they pass through the column between the beads and flow out into the collection tubes or fractions early. Molecules that are the size of the tunnels or smaller will flow through the column more slowley and elute in later fractions because they enter the tunnels. It is important, if using this technique, to have an idea of how large the molecule is you are trying to purify.
You have a protein of interest that you need to purify and have found that this protein binds strongly to GTP. What type of chromatography would be best to purify this protein? Why? How would you do this?
To purify a protein that binds strongly to GTP, you would use affinity chromatography because it will be easy to separate this protein from other components that do not bind specifically to GTP. You would use a column with GTP covalently linked to the support beads and add the sample. The sample will bind the column and all other molecules will flow through. You then remove or elute the protein by adding extra GTP in some media to the column, which will displace your protein and allow it to flow out.
What molecular characteristics determine separation by HPLC?
The molecular characteristic that determines separation by HPLC, or high pressure liquid chromatography, is the polarity of the molecule you are trying to purify.
What is a his‑tagged protein? How can this type of protein be purified?
A his‑tagged protein is a recombinant protein that has a histadine tag attached to it to purify it on a column. This is achieved by adding the nucleotides needed for 6 histadine residues when cloning a gene into a plasmid. This type of protein can be easily purified using a histadine column, by applying the sample to a protein with a support of cobalt or nickel. The histadine residues will allow the protein of interest to bind the column, while all other proteins in the lysate will flow through. The his‑tagged protein can be released from the support by adding imidazole, which will displace the protein and allow it to flow through the column and be collected in a later fraction.
What is the purpose of electrophoresis? What types of molecules are subjected to agarose gel electrophoresis? To SDS‑PAGE?
The purpose of electrophoresis is to separate DNA, RNA, or proteins by size using an electrical current.
DNA and RNA are separated by agarose gel electrophoresis, and proteins are separated using SDS‑PAGE.
Why is agarose used in agarose gel electrophoresis, and acrylamide used in SDS‑PAGE?
Agarose is used in agarose gel electrophoresis, because agarose is a gel‑like matrix with openings that allow DNA and RNA to pass, and the openings in the matrix of agarose are too large to use for proteins. Proteins are smaller, globular, and vary in their charge, so acrylamide is used for SDS‑PAGE. Acrylamide is a matrix that can be adjusted to separate larger or smaller proteins, and sodium dodecyl sulfate (SDS), is a detergent that denatures the proteins and coats them with a negative charge so they can be separate by size.
What is the advantage of 2D gel electrophoresis?
The advantage of 2D gel electrophoresis is that proteins can be separated by charge and size. This provides more separation of a mixture of proteins, so that each spot on the gel should be only one unique protein.
The spots can be cut out and sent for Mass Spectrometry analysis to identify the individual protein.
You have a tissue sample of a wound and want to study what proteins are involved in wound healing. What type of electrophoresis would be best to identify one protein involved? What additional technique would be needed to identify that protein?
The best method to identify a protein involved in wound healing would be to use 2D gel electrophoresis, so that you can separate individual proteins. Mass spectrometry would also be needed to find the sequence and identity of the protein.
What is the purpose of blotting? Differentiate between Southern, Northern, and Western blotting.
The purpose of blotting is to identify one DNA or RNA sequence or one protein from a mixture. This is achieved by using a probe or an antibody that will bind only that molecule. Southern blots are used for DNA, Northern blots for RNA, and Western blots for protein.
What is the typical probe used for Western blotting? How is the binding visualized?
The typical probe used for Western blotting is an antibody specific to the protein of interest. The antibody is bound with a second antibody that is conjugated to a compound, so that the binding can be visualized.
Suppose you want to screen a patient for a mutation in the p53 gene, which is responsible for tumour suppression and control of malignant growth. Most people with cancer have a mutation in the p53 gene. What blotting method would you use to determine if a patient has a mutation in p53 and why?
To determine if a patient has a mutation in the p53 gene, you would use Southern blotting, because this is the method used for identifying a DNA sequence of interest or a change in a DNA sequence.
What is the basic principle behind DNA microarrays?
The basic principle behind DNA microarrays is that they can provide a lot of information about what genes are being transcribed in a cell under a particular condition. DNA microarrays provide an entire picture of what is going on in a certain condition by demonstrating what genes are expressed in a particular environment or tissue at a given time.
Name and describe two applications of DNA microarrays.
Two applications of DNA microarrays are:
Gene expression profiling: expression levels of thousands of genes are simultaneously monitored to study the effects of treatments, diseases, developmental stages.
Comparative genomic hybridization: compares the genomes of different cells or closely related organisms to see if there are differences in what genes they possess.
What is the function of DNA ligase in constructing rDNAs?
DNA ligase is used in the construction of rDNAs to join the digested “sticky” or matching ends together so the DNA sequence of interest can be inserted into a plasmid.
Name one rDNA that is in use, and discuss why the rDNA form is beneficial.
One rDNA in use is recombinant human insulin. The recombinant form is beneficial, because animals do not have to be used as a source of insulin for treatment of insulin‑dependent diabetes. The recombinant form is a harmless way of having abundant insulin available at a lower cost.
What is the purpose of PCR?
The purpose of PCR is to obtain multiple (millions) of copies of a gene or a DNA sequence, so that it can be visualized or used for cloning.
Why does the DNA polymerase used in PCR need to be heat‑stable?
The DNA polymerase used in PCR has to be heat‑stable, because the high temperature used to separate DNA strands during PCR (so that they can be replicated) can cause an enzyme to degrade.
Name one application of PCR.
DNA cloning for expression or sequencing.
Others are DNA‑based phylogeny (functional analysis of genes),
DNA fingerprinting (forensic science and paternity testing),
diagnosis of hereditary diseases, and
detection and diagnosis of infectious diseases).
What is Lac Z Blue‑White screening used for?
Lac Z Blue‑White screening is used to identify clones that contain the correct DNA sequence inserted into a plasmid. This is because, when the DNA is cloned into a plasmid containing the β‑galactosidase gene, properly adding the transformed bacteria with the plasmid to media containing X‑gal will allow identification of the clones with the properly inserted DNA sequence, because those clones will be able to utilize the lactose in the agar plate and the colony will turn blue.
What is the product of reverse transcription? What does the enzyme reverse transcriptase do?
The product of reverse transcription is a cDNA or complementary DNA sequence. The enzyme reverse transcriptase converts RNA to a DNA sequence.
