Transcript Introduction to Vectors

Duckweed: Sequencing the Genome
Contains living cells that are producing proteins.
DNA RNA Protein
Through "Molecular Cloning“ or
"Genetic Engineering“ or
"Recombinant DNA Technology“
we can sequence the DNA of
Duckweed
DNA  RNA  Protein
DNA  RNA  Protein
AAAAA
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
Plasmids
• Circular DNA molecules found in
bacteria
• Replicated by the host’s 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
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
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
Cloning Scheme
After isolating mRNA, convert mRNA to cDNA with rev
transcriptase. Ligate insert into plasmid.
Wolffia DNA
Digest
Ligate
Amplify and Prep
Transform…
plasmid into bacteria
How do you find the
bacteria with the
plasmids?
Transform…
plasmid into bacteria
ampicillin
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
Plasmid cloning vector
pDNR-Lib
The cDNA insert is cloned into
the SfiI sites
cDNA Insert
MCS A
MCS B
p. 1-4
Construction of a
cDNA library
p. 1-6
Differences between a genomic and cDNA library
Genomic Library
Promoters
Introns
Intergenic
Non-expressed genes
cDNA Library
Expressed genes
Transcription start sites
Open reading frames (ORFs)
Splice points
p.1-7
Construction and analysis
of a genomic DNA library
p. 1-5
Synthesis of cDNA from mRNA
p. 1-8
SfiI digestion sites of pDNR-Lib
p. 1-9
Cloning W.a. cDNA fragments into the
pDNR-Lib polylinker
p. 1-10
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
Naming your clones
Your initials
Year
20AV12.09
School #
Clone #
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