Genomics- #8 Classic Cloning.txt

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Genomics- #8 Classic Cloning.txt
2012-02-10 11:37:20
HUSOP Gen EXAM1 Classic Cloning

Questions from Genomics Classic Cloning
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  1. the names of the twins of Argos are:
    Kleobis and Biton
  2. ___________ is known as the process of introducing a cloning or expression vector into a host cell.
    Transfection of Cells
  3. 3 methods of Transfection of Cells
    • 1) Viral
    • 2) Chemical
    • 3) biophysical
  4. chemical transfection involes
    neutralize electric charge on DNA
  5. Biophysical transfection involes
  6. coating DNA with CaPO4 allows the DNA to precipitate via ________
    pinocytosis (cell drinking)
  7. DNA __________tends to be more protective and shielding of DNA
  8. The use of _________ _______ is preferred for putting a vector in a mammalian cell
    artificial lipids
  9. __________ uses high field potential in short bursts
  10. the first step to classical cloning is to
    isolate total RNA from a tissue and then purify mRNA from it
  11. total RNA contains ______(3)
    • mRNA,
    • rRNA,
    • snRNA
  12. _______ _____ is the way to purify mRNA
    affinity chromatography
  13. _____RNA has a poly dA tail on it that tags it as funtional
  14. adjusting the _______maximizes the fraction interation
    salt concentration
  15. to convert mRNA to ssDNA use
    reverse transcriptase and deoxynucleotides
  16. to convert ssDNA to dsDNA use
    DNA dependent DNA polymerase (deoxynucleotides)
  17. In order to insert the appropriate dsDNA into the vector it is opened by digestion with an appropriate
    restriction nuclease
  18. in order to insert the cloning vector correctly into DNA, the DNA must open in
    a selectable marker domain
  19. T/F a different restriction nuclease must be used to digest dsDNA than that used to open it
  20. ________ bonds are made by adding DNA ligase
  21. The most widely used method to introduce the vector into E. coli is by the
    CaPO4 precipitation method
  22. DNA is added in a _____solution along with a HPO4= buffer.
  23. The DNA co-precipitates with the _____ onto the E. coli cell walls where it is taken up into the cells.
  24. the method of introducing vectors into host bacteria is called
    Transfection of Cells
  25. streaking E.coli in antibiotic agar is a method of _____ & ______
    isolation and selection
  26. T/F Transfected bacteria die in the prescense of its non-disrupted selectable antibiotic
  27. _______ ____ _______Refers to the ability of nucleic acid polymers to base pair and bind to each other.
    Nucleic Acid Hybridization
  28. This is an example of solid phase hybridization.
    Southern Blot
  29. Hybridization occurs between __________&__________
    • cellular DNA
    • DNA probe (specific sequence of DNA)
  30. Southern Blot Step 1. _________ DNA through an agarose slab gel
  31. SB Step 2. ________ the DNA to a solid support
  32. SB Step 3. Hybridize DNA on solid support to a
    DNA probe labeled with 32P
  33. SB Step 4. ________ _________ to remove non-bound DNA probe.
    Wash extensively
  34. SB Step 5. Detect where DNA probe is ________ by exposing to X-ray film.
  35. exposures on gel plates likely indicate _____
    DNA complementary to the probe
  36. Uses of a Southern Blot (2)
    • clinical gentetics
    • viral infection confirmation
  37. SB clinical genetics can detect (2)
    • size changes
    • base pair mutations
  38. SB viral infection confirmation detects
    the presence of viral gene sequences in human DNA
  39. Northern blot is similar to southern blot except
    RNA is seperated and supported on a solid (electorphoresis gel)
  40. Northern Blot starts with ____, while Southern Blot starts with ______
    • RNA,
    • DNA
  41. NB clinical genetics determines if�
    abnormal DNA is expressed in mRNA
  42. NB viral confirmation determines...
    the presence of viral gene products
  43. ______ ________ is used to examine the complement of a set of gene products
    Individual phenotyping
  44. In this approach, the hybridization of RNA to a radiolabeled probe occurs in solution.
    Solution Hydridization of mRNA
  45. Solution Hydridization of mRNA advantages (2)
    • increased sensitivy frm enhanced probe acces
    • multiple probes in a single tube
  46. SH Step 1. In a tube, _____ _____ _____ is mixed with a 32P-labeled RNA probe.
    total cellular RNA
  47. SH Step 2. Add _____ & _________ to the tube to degrade all single stranded RNA
    • RNase A
    • RNase T1
  48. dsRNA is protected from degredation during solution hybridization,so this is called a�
    RNA Protection Assay