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22 Cards in this Set
- Front
- Back
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Which technique depends on the ability to alternately denature dsDNA molecules and hybridize complementary single strands in a controlled fashion?
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PCR
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The annealing temperature is generally how many degrees lower that Tm?
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3-4
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PCR begins with the _________ of DNA into ______ ______?
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Denaturation, single strands
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With each cycle in PCR, the number of copies of the sequence between the primer sites is increased by what?
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Doubled
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The duration of extension for PCR is how long per 1kb?
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1 minute
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What is the easiest way to clone a sequence of interest if the sequence is known?
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PCR
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What is the first step in Southern blotting?
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Cleavage of DNA by restriction endonucleases.
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How is the fragment of interest identified in Southern blotting when there are so many different fragments of the same length?
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It is identified by its ability to hybridize to a specific DNA probe.
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In Southern blotting, what are the restriction fragments present on the gel transferred to?
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Nitrocellulose filter or nylon membrane by blotting.
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Expression of a particular gene is followed by assaying for the corresponding mRNA by which technique?
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Northern blotting
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G-CSF protein expression in E. coli can be induced how?
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Plasmid transformation of desired protein DNA sequence next to lac promoter in E. coli. Transcription from lac promoter will occur giving the desired protein.
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Which type of transfection involves integration of the vector into the host genome?
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Stable
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How does transient transfection work?
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Plasmid vectors containing an origin of replication enter a mammalian cell. The origin of rep allows it to replicate efficiently, generating numerous plasmids from which the protein is expressed. During cell division, however the plasmids are not segregated into both daughter cells, and over time some cells will not contain a plasmid. MAIN POINT - not integrated into host chromosome
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How does stable transfection occur?
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The introduced vector integrates into the host chromosome and thus "tranformes" it. This means that these cells will continue to produce the protein as long as the culture is maintained.
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How do tags such as GST and 6xHIS work?
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You combine your gene for your target protein with another (GST, 6xHIS) that can be bound or anchored to something to easily purify it.
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How can PCR be used to create a construct for disrupting a target gene?
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The two primers designed for this purpose contain a sequence of about 20 nt homologous to one end of the target gene. The resulting amplified construct comprises the selectable marker.
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T/F: It is possible to knock out more than one chromosome at a time?
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False
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T/F yeast and mammals both are good at homologous recombination?
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False, yeast are, mammals are not.
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What is inactivation by dominant negative allele?
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Create a protein that will affect the protein of interest, thus inactivating it.
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Explain deletion detection of nested primers.
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Run PCR with two primers on area of interest. then run second PCR with primers closer together inside the sequence. Then run on the gel and examine for the difference.
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What tandem repeats are used for DNA fingerprinting?
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RFLP and SSR
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What is RFLP and what is it used for?
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Restriction fragment linked polymorphism.They are variations in homologous DNA sequences. Can be used for DNA fingerprinting.
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