Steps of Recombinant DNA Technology

22 terms by happyflipican 

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First Step

Isolation of plasma DNa & DNA containing gene of interest

2nd Step

Gene inserted into plasmid

3rd Step

Plasmid put into bacterial cell

4th Step

Cells cloned with gene of interest

Fifth Step

Identification of desired clone

Applications

pest resistance, clean oil spills, when contain antifreeze, vaccine patterns

Central Dogma

DNA, RNA, protein, trait

Uses for Molecular Biology

Curing cancer, heart disease, Alzheimer's disease
Genetically manipulate corn for high yield & enhanced flavor
Genetically manipulate tomatoes for slower ripening
Identify criminals cases- murder or rape
DNA fingerprinting

Cytocrom C

important electron carrier in respiratory pathaway, found in all aerobic eukaryotes

Restriction endonucleases

enzymes that have been purified from a different species of bacteria and restriction enzyme

Restricting/digesting

Cutting requires energy in form of ATP, involves physical cleaving of chemical bonds,
specific recognition, sites where cuts occur and are palodromic

Plasmid

Lab, Puc19 and E.Coli, relatively small extrocromisonal, circular molecule of DNA, found in bacteria and yeast
Puc19 used for cloning because of size and DNA sequence

Agacrose gel

Used to separate and sort fragments of DNA by size only, DNA fragments placed in gel, electric current runs through matrix, fragments move at different rates depending on protein's size and charge

Steps for cloning a gene and bacterial plasmid

1. Isolate DNA from two sources
2. Cut both DNA with some restriction enzyme
3. Mix DNA they join by base pairing
4. Add DNA ligase to bond covalently
5. Put plasmid into bacterium by transformation
6. Identify cells containing recombinant plasmid by ability to grow in presence of ampR and tetR
7. Clone selected bacteria

Methaline Blue

Used to stain gel after DNA samples were ran, allow visualization of discrete DNA bands on gel

kilobase

size of DNA fragment is measured in nucleotide bases, 1000 base pairs= 1 Kb

restriction enzyme

recognize specific DNA sequence whenever it occurs in a DNA molecule and cut DNA near site, each restriction enzyme is named for the species of bacteria from which is located & isolated; Bacteria uses restriction enzymes to recognize & metabolize foreign DNA, assigned units based on amount required to digest 1 microgram of DNA in 1 hour

Ava2

restriction enzyme found in arabra bariabilis

Pvu2

restriction enzyme found protens vulgans that cuts

Molecular weight markers

Used to determine size of DNa fragments & Are of known size w/ which to compare results

Tris-borate-EDTA (TBE)

buffer used for agarose gels electrophoresis in analysis of DNA products resulting from PCR amplification, using wrong concentration of TBE can result in gel and buffer over heating

Ethidium bromide

Used to stain gel, toxic binds the DNA & fluroesces under ultraviolet (UV) light, bigger DNA, alternative staining technique

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