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Steps of Recombinant DNA Technology

STUDY
PLAY
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