Lab 6: Molecular Biology
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small circular DNA molecules that carry genetic info; some are extrachromosomal; they exist separately from the chromosome; some replicate only when the bacterial chromosome replications; others autonomously and often occur in as man as 10-200 copies within a single bacterial chromosome
In nature, genes can be transferred between bacteria in three ways: __, __, __
- bacterial transformation
mating process during which genetic material is transferred from one bacterium to anoter of a different mating type
requires the presence of a virus ot act as a vector (carrier) to transfoer small pieces of DNA from one bacterium to another
involves transfer of genetic info into a cell by direct uptake of the DNA
Bacteria can take up DNA only during te period at the end of logarithmic growth, called __.
__ is expressed as the number of antibiotic-resistant colonies per microgram of pAMP
__ are found in prokaryotic organisms that cut up foreign pieces of DNA at specific recognition sites; recognize specifc DNA sequences in double-stranded DNA and digest hte DNA at these sites; the result is production of fragments of DNA of various lengths
What is mobility or speed influenced by when any molecule enters an electrical field?
charge of the molecule, strength of the electrical field, size and shape of the molecule, density of the gel through which it is migrating
In agarose gel, the the smaller the DNA, the __ it migrates through the gel.
faster and farther
In the graph of the gel electrophoresis, what is the independent and depnedent variable?
- dependent: number of base pairs (y- axis)
- independent: migration distance (x- axis)
Effect of voltage used on electrophoresis?
- higher voltage: DNA move faster
- lower: DNA move slower
Effect of Running time on electrophoresis?
- longer time: DNA would've ran off the gel
- shorter time: not far enough and would have caused the bands to not separate enough, being merged and unable to be read
Effect of Amount of DNA used on electrophoresis?
more DNA: more distinct bands because they would have all traveled the same distance if the same fragment size
Effect of Reversal of Polarity on electrophoresis?
the DNA would've moved backward into the wells
What is a plasmid? How are plasmids used in genetic engineering?
- plasmids are small circular molecules found in prokaryotes that carry their own gentic info; can transfer genes and act as a vector
- - used because they can introduce certain favorable genes into bacteria or other genome,causing the organism to express that gene, for example ampR+
What are restriction enzymes? How do they work? What are recognition sites?
- enzymes found in prokaryotes that cut foreign DNA into fragments; they cut at specific recognition sites, creating blunt or sticky ends. They cut the bonds of DNA.
- - Recognition sites are specific DNA sequences that are recognized by the endonucleases and cut at these sites.
What is the source of restriction enzmyes? What is their function in nature.
- prokaryotes and bacteria
- to protect the bacterial cell from injection of foreig DNA by cutting up the DNA into fragments
Describe the function of electricity and agarose gel in electrophoresis?
- electricity: pulls the DNA toward the opposite end of hte gel, allowing it to separate. It determines the distance of migration.
- gel: allows the trail and separation of DNA to be recorded; liquids and solids cant do this; the gel allows us to see the fragments to be seen once the dye is put in.
What are the functions of loading dye in electrophoresis? How can DNA be prepared for visualization?
- the dye: allows DNA to become more visible and enables us to record the data and determine the distance migrated and possibly hte base pair size and the number of bands.
- visualization: DNA can be subject to a fluorescent dye that binds the gel into a series of bands.
How can a mutation that alters a recognitionsite be detected by gel electrophoresis?
By using hte same restriction enzyme on the same DNA genomes, the restriction enzyme will produce different fragments because of the mutations. THis will cause a differnet pattern in electrophoresis and different fragment sizes.
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