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General Biology: Recombinant Plasmid Cloning and Extraction

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이 집합의 용어 (27)

  • What is the purpose of creating recombinant plasmids in this lab?
    To clone and analyze fragments of the yeast genome by inserting yeast DNA into a plasmid vector and transforming E. coli cells.
  • What is a plasmid vector?
    A small circular DNA molecule used to carry foreign DNA fragments into a host cell for cloning.
  • What key components does the pBluescript (pBS) vector contain?
    An origin of replication, an ampicillin resistance gene (ampR), and the LacZ reporter gene.
  • What is the function of the origin of replication in the plasmid?
    It allows the plasmid to be replicated independently within the bacterial host.
  • Why is the ampicillin resistance gene important?
    It acts as a selectable marker, allowing only bacteria with the plasmid to grow on ampicillin-containing media.
  • How does the LacZ gene function as a reporter gene?
    It encodes β-galactosidase, which cleaves X-gal to produce blue colonies if intact; disruption by insert DNA results in white colonies.
  • What does a white colony on an ampicillin/X-gal plate indicate?
    The colony contains a plasmid with a yeast genomic DNA insert disrupting the LacZ gene.
  • What role does EcoRI play in plasmid construction?
    EcoRI is a restriction enzyme that cuts both the yeast genomic DNA and pBS vector to create compatible sticky ends for ligation.
  • Why is the pBS vector treated with phosphatase before ligation?
    To remove 5' phosphates from vector ends, preventing self-ligation and promoting insertion of yeast DNA.
  • What enzyme catalyzes the formation of covalent bonds between DNA fragments during ligation?
    DNA ligase.
  • What is transformation in this context?
    The process of introducing recombinant plasmid DNA into competent E. coli cells.
  • Why are E. coli cells described as 'competent'?
    They have been treated to allow uptake of foreign DNA.
  • What is the purpose of the mini-prep procedure?
    To extract and purify recombinant plasmid DNA from E. coli cells.
  • What is the role of EDTA in the plasmid extraction resuspension step?
    It removes Mg2+ ions, inactivating nucleases that could degrade DNA.
  • Why is RNase A added during plasmid extraction?
    To degrade RNA and prevent contamination of the plasmid DNA sample.
  • What does SDS and NaOH do during the lysis step?
    SDS disrupts membranes and denatures proteins; NaOH denatures DNA and degrades the cell wall.
  • How does neutralization separate plasmid DNA from chromosomal DNA?
    Plasmid DNA renatures and remains soluble; large chromosomal DNA becomes tangled and precipitates with proteins.
  • What is the function of the spin column with silica oxide matrix?
    It binds plasmid DNA under chaotropic salt conditions, allowing impurities to be washed away.
  • What reagent in the neutralization buffer helps plasmid DNA bind to the silica matrix?
    Guanidine hydrochloride, a chaotropic salt.
  • How is plasmid DNA finally recovered after washing the spin column?
    By eluting with pure water to rehydrate and collect the plasmid DNA.
  • What is the significance of the multiple cloning site (MCS) in the LacZ gene?
    It contains restriction sites for enzymes like EcoRI, allowing insertion of foreign DNA without disrupting the entire gene.
  • Why do recombinant plasmids have two EcoRI sites after ligation?
    Because both the vector and insert DNA were cut with EcoRI, creating compatible sticky ends that rejoin.
  • What is the expected size range of yeast genomic EcoRI fragments in the library?
    From a few base pairs up to over 10,000 base pairs, with an average around 4 kb.
  • Why is it important to have a control plate without ampicillin during transformation?
    To verify cell viability and survival independent of transformation.
  • What does the presence of blue colonies on ampicillin/X-gal plates indicate?
    Cells took up the pBS vector without an insert, so LacZ is intact and β-galactosidase is produced.
  • How many copies of plasmid DNA can each E. coli cell contain?
    Up to about 500 copies per cell due to the origin of replication.
  • What is the main difference between nucleic acid isolation in Lab 2 and plasmid isolation in Lab 5?
    Lab 5 isolates only plasmid DNA using selective binding and precipitation, while Lab 2 isolated total nucleic acids based on solubility.