Experiment 1, Day 1 Flashcards
Recombinant DNA
2 or more fragments of DNA that were combined in the lab.
Cloning (biotechnology)
Making exact copies of DNA fragment, cells or organisms
Gene of Interest
Gene that encodes the product we want to produce.
Plasmids
Smaller circular DNA strand in the cytoplasm of a bacterium that is able to replicate independently of the chromosomes.
Vector
An organism ( such as an insect) that transmits a pathogen.
Restriction Enzymes
Enzymes that cut specific segments of DNA
Natural Plasmids
Naturally occurring small circular molecules of DNA that exist in bacteria
Commercial Vector and Features:
Natural plasmids that have been modified in the lab to facilitate inserting new DNA fragments
- Ori
- selectable marker
- multiple cloning site
How can we make human rote is (e.g, insulin) using transgenic bacteria?
- Transgenic bacteria are manipulated in a lab.
- Scientists can cut specific genes from one organism and paste the genes as part of the DNA of another organism.
Selective Breeding
The process by which humans bred other animals and plants for particular traits.
Ex: dog breeds, all produce and meat
How do you make recombinant DNA?
- Cutting the desired DNA by restriction sites
- Amplifying the gene copies by PCR
- Inserting the genes into the vectors
- Transferring the vectors into host organism
- Obtaining the products of recombinant genes
How do restriction enzymes work? What are their properties?
They recognize and bind to specific sequences of DNA, called restriction sites. Each restriction enzyme recognizes just one or a few sites. When it finds its target sequence, a R.E. Will make a double-stranded cut in the DNA molecule, producing ends with single-stranded DNA overhangs or blunt ends.
Steps for bacterial Transformation
- Specially prepared bacteria are mixed with DNA, from a ligation.
- Heat shock bacteria, which causes some of them to take up a plasmid
- Plasmids used in cloning contain an antibiotic resistance gene.
- Bacteria without a plasmid die, each bacterium with a plasmid gives rise to a cluster of. Identical, plasmid-containing bacteria called a colony
Competent cells and chemically competent
Bacterial cells that have been specially treated to transform efficiently.
Are treated with a buffer that contains CaCl2 and other salts that disrupt the cell membrane creating “holes”. That allow the plasmids to pass into the cell.
