Lab Day 1 Exp 1 Flashcards
why are microbes beneficial/harmful for water quality
microbes are beneficial as they can detoxify pollutants
microbes are harmful as they can be a source of water contaminants
what are the amplified PCR products
they are amplified regions of the small ribosomal subunits from various microbes, this region is hypervariable
what is our goal
isolate and analyze individual PCR products
what is the first step of the procedure
cut open the circular plasmid with a restriction enzyme
once we have cut open the circular plasmid, what is the next step
ligate the foreign DNA into a plasmid vector
when we have the recombinant DNA molecule, what is the next step
we will introduce it to the E-coli cells via transformation
what is a competent cell? How do we make E-coli competent?
a cell that can take up plasmids
we can make E-coli more competent by incubating it in CaCl2 and a heat shock
after the bacterial host has taken up the plasmid, what do we do
we plate the bacteria onto an antibiotic plate
why do we use antibiotic plates? what would happen if we didnt
we use antibiotic plates because the plasmids have an antibiotic-resistant selectable marker that makes them resistant to the antibiotic. Only transformed cells will have this resistant gene, thus they are the only ones that will grow
if we didnt use this plate, all the bacteria including the nontransformed bacteria would grow
after we select for transformants with antibiotic plate, what is the next step
we will purify the plasmid DNA and then digest it with different restriction enzymes which will cut differently based on sequence
then we will run a gel electrophoresis to allow visualization and analysis of the plasmids cut with the enzymes
what is the purpose of gel electrophoresis
to visualize and analyze the plasmids cut with the restriction enzymes
what will this process overall allow us to do with the microbacteria
it will allow us to draw conclusions about sequence similar and dissimilar from ribosomal gene regions from different microbes
what is molecular cloning
it is where we will insert a DNA fragment into a vector and recover the plasmid from a colony of bacterial cells that are identical with respect to their DNA as they were derived from a single ancestral cell
what is a plasmid
a plasmid is a small double stranded circle of DNA that is naturally carried by some bacteria in addition to its larger circular genome
why is it important for plasmids to have an origin of replication and be self replicating
this is important because it allows them to be replicated along with regular bacterial DNA and not be lost
what is transformation
transformation is the process by which bacteria spontaneously take up a plasmid DNA from the surrounding environment
what are the three features of plasmids for molecular cloning
origin of replication
cloning site
selectable marker
what is a cloning site
a cloning sites are the recognition sites for restriction enzymes to cut the plasmid
what if we didn’t have a cloning site?
there would be no place to cut the plasmid and we could not insert the foreign DNA
what did we use as our selectable marker? what would happen if we didn’t have the marker?
we used a kanamycin resistant gene in the plasmid. If we didn’t use this, we wouldn’t have a way of identifying which cells had taken up the plasmid via transformation
what forms the double stranded break in the DNA
restriction enzyme
what seals the DNA back together? How?
DNA ligase connects the 3’ OH to a phosphate group at the 5’ at the next nucleotide through a phosphodiester bond
what is a sticky end
a sticky end is formed by a restriction enyzme that cuts at different locations
what is a blunt end
it is formed by a restriction enzyme that cuts at the same location
can ligase only seal sticky ends?
no it can seal both blunt and sticky ends
what does CaCl2 and heat shock do to the membrane of the ecoli?
it weakens the membrane and allows the plasmid to enter through the membrane
