duckweed: sequencing the genome

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Duckweed: Sequencing the Genome Contains living cells that are producing proteins. DNA RNA Protein

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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. - PowerPoint PPT Presentation

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Page 1: Duckweed: Sequencing the Genome

Duckweed: Sequencing the Genome

Contains living cells that are producing proteins. DNA RNA Protein

Page 2: Duckweed: Sequencing the Genome

Through "Molecular Cloning“ or "Genetic Engineering“ or

"Recombinant DNA Technology“ we can sequence

the DNA of Duckweed

DNA RNA Protein

Page 3: Duckweed: Sequencing the Genome

DNA RNA Protein

AA

AA

A

Page 4: Duckweed: Sequencing the Genome

Purification of mRNA

p. 1-8

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)

Page 5: Duckweed: Sequencing the Genome

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

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

Page 6: Duckweed: Sequencing the Genome

VectorsIn 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

Page 7: Duckweed: Sequencing the Genome

• 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.

Plasmid Engineering

p. 1-2

Page 8: Duckweed: Sequencing the Genome

Cloning Scheme

Digest Ligate Amplify and Prep

Wolffia DNA

After isolating mRNA, convert mRNA to cDNA with rev transcriptase. Ligate insert into plasmid.

Page 9: Duckweed: Sequencing the Genome

Transform…plasmid into bacteria

How do you find the bacteria with the plasmids?

Page 10: Duckweed: Sequencing the Genome

Transform…plasmid into bacteria

ampicillin

Page 11: Duckweed: Sequencing the Genome

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

Page 12: Duckweed: Sequencing the Genome

Plasmid cloning vector pDNR-Lib

The cDNA insert is cloned into the SfiI sites

p. 1-4

cDNA Insert

MCS A MCS B

Page 13: Duckweed: Sequencing the Genome

Construction of a cDNA library

p. 1-6

Page 14: Duckweed: Sequencing the Genome

Differences between a genomic and cDNA library

p.1-7

Genomic LibraryPromotersIntronsIntergenicNon-expressed genes

cDNA LibraryExpressed genesTranscription start sitesOpen reading frames (ORFs)Splice points

Page 15: Duckweed: Sequencing the Genome

Construction and analysis of a genomic DNA library

p. 1-5

Page 16: Duckweed: Sequencing the Genome

Synthesis of cDNA from mRNA

p. 1-8

Page 17: Duckweed: Sequencing the Genome

SfiI digestion sites of pDNR-Lib

p. 1-9

Page 18: Duckweed: Sequencing the Genome

p. 1-10

Cloning W.a. cDNA fragments into the pDNR-Lib polylinker

Page 19: Duckweed: Sequencing the Genome

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

Page 20: Duckweed: Sequencing the Genome

20AV12.09

Naming your clones

YearYour initials

School # Clone #

School #1. Bayonne3. Colonia4. East Brunswick5. High Point6. Hillsborough7. James Caldwell8. JFK Memorial9. JP Stevens10. Monmouth11. Montville12. New Brunswick13. Pascack Hills14. Pascack Valley15. Rutgers Prep.16. Somerville17. The Pingry School18. Watchung Hills19. West Windsor-Plains.20. Rutgers University21. Liberty24. Lynbrook28. Roland Park Country29. Archbishop Curley30. Largo31. DuVal32. Great Mills33. McDonogh34. Science & Math. Acad.35. Walter Johnson36. North County37. Thurgood Marshall38. Hackettstown40. Bordentown