Duckweed: Sequencing the Genome

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

AA

AA

A

Purification of mRNA

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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 200-500 copies/cell

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

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.

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• 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

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Cloning Scheme

Digest Ligate 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.

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Plasmid cloning vector pDNR-Lib

The cDNA insert is cloned into the SfiI sites

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cDNA Insert

MCS A MCS B

Construction of a cDNA library

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Differences between a genomic and cDNA library

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Genomic LibraryPromotersIntronsIntergenicNon-expressed genes

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

Construction and analysis of a genomic DNA library

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Synthesis of cDNA from mRNA

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SfiI digestion sites of pDNR-Lib

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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.

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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