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

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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)

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

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

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

Cloning Scheme DigestLigate Amplify and Prep Wolffia DNA After isolating mRNA, convert mRNA to cDNA with rev transcriptase. Ligate insert into plasmid.

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 p. 1-4 cDNA Insert MCS A MCS B

Construction of a cDNA library p. 1-6

Differences between a genomic and cDNA library p.1-7 Genomic Library Promoters Introns Intergenic Non-expressed genes cDNA Library Expressed genes Transcription start sites Open reading frames (ORFs) Splice points

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

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

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

20AV12.09 Naming your clones YearYour initials 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