Introduction to Vectors

1
Duckweed Sequencing the Genome
Contains living cells that are producing
proteins. DNA? RNA? Protein
2
Through "Molecular Cloning or "Genetic
Engineering or "Recombinant DNA Technology we
can sequence the DNA of Duckweed
DNA ? RNA ? Protein
3
DNA ? RNA ? Protein
4
Purification of mRNA
Collect and grind up plants in mild denaturing
solution
Spin out debris (Tissue, membranes, etc)
Treat with DNAse (removes DNA)
Treat with Phenol (removes protein)
p. 1-8
5
Plasmids

• Circular DNA molecules found in bacteria
• Replicated by the hosts machinery independently
  of the genome. This is accomplished by a sequence
  on the plasmid called ori, for origin of
  replication.
• Some plasmids are present in E. coli at 200-500
  copies/cell

The most common bacterial plasmids are members of
the pUC series- Waksman Chair, J. Messing
p. 1-1
6
Vectors

• In order to study a DNA fragment (e.g., a gene),
  it needs to be amplified and eventually purified.
• Accomplished by cloning the DNA into a vector.
• This vector is a plasmid is small, circular DNA
  molecule that replicates inside a bacterium such
  as Escherichia coli.

p. 1-1
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Plasmid Engineering

• Plasmids also contain selectable markers.
• Genes encoding proteins which provide a selection
  for rapidly and easily finding bacteria
  containing the plasmid.
• Provide resistance to an antibiotic (ampicillin,
  kanamycin, tetracycline, chloramphenicol, etc.).
• Thus, bacteria will grow on medium containing
  these antibiotics only if the bacteria contain a
  plasmid with the appropriate selectable marker.

p. 1-2
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Cloning Scheme
After isolating mRNA, convert mRNA to cDNA with
rev transcriptase. Ligate insert into plasmid.
Wolffia DNA
Digest
Ligate
Amplify and Prep
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Transform plasmid into bacteria
How do you find the bacteria with the plasmids?
10
Transform plasmid into bacteria
ampicillin
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DNA Libraries

• DNA library - a random collection of DNA
  fragments from an organism cloned into a vector
• Ideally contains at least one copy of every DNA
  sequence.
• Easily maintained in the laboratory
• Can be manipulated in various ways to facilitate
  the isolation of a DNA fragment of interest to a
  scientist.
• Numerous types of libraries exist for various
  organisms - Genomic and cDNA.

p. 1-5
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Plasmid cloning vector pDNR-LibThe cDNA insert
is cloned into the SfiI sites
cDNA Insert
MCS B
MCS A
p. 1-4
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Construction of a cDNA library
p. 1-6
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Differences between a genomic and cDNA library
Genomic Library Promoters Introns Intergenic Non-e
xpressed genes
cDNA Library Expressed genes Transcription start
sites Open reading frames (ORFs) Splice points
p.1-7
15
Construction and analysis of a genomic DNA
library
p. 1-5
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Synthesis of cDNA from mRNA
p. 1-8
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SfiI digestion sites of pDNR-Lib
p. 1-9
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Cloning W.a. cDNA fragments into the pDNR-Lib
polylinker
p. 1-10
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Essential components of minipreps

• Gentle lysis step to break open the cells and
  release the plasmid DNA into solution.
• Cell debris and chromosomal DNA of the bacteria
  is pelleted during the centrifugation.
• Plasmid DNA remains behind in the clear
  nonpelleted fraction (the nonpelleted solution
  left after centrifugation is known as the
  supernatant).
• Subsequent steps are then performed on the
  supernatant to remove contaminating RNA and
  proteins from the plasmid DNA.

p. 1-11
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School 1. Bayonne 3. Colonia 4. East
Brunswick 5. High Point 6. Hillsborough 7. James
Caldwell 8. JFK Memorial 9. JP Stevens 10.
Monmouth 11. Montville 12. New Brunswick 13.
Pascack Hills 14. Pascack Valley 15. Rutgers
Prep. 16. Somerville 17. The Pingry School 18.
Watchung Hills 19. West Windsor-Plains. 20.
Rutgers University 21. Liberty 24. Lynbrook 28.
Roland Park Country 29. Archbishop Curley 30.
Largo 31. DuVal 32. Great Mills 33. McDonogh 34.
Science Math. Acad. 35. Walter Johnson 36.
North County 37. Thurgood Marshall 38.
Hackettstown 40. Bordentown
Naming your clones
Year
Your initials
20AV12.09
School
Clone