agar plate
Cells are often spread out onto plates (see plating) made of a
gelatinous material called agar. The agar media often contains nutrients
for the cells, and in our case also contains antibiotics.
ampicillin
This is an antibiotic which normally can kill E. coli cells.
ampicillin resistance gene
This gene is found in the pAMP plasmid; the protein which this gene codes
for allows an E. coli cell to survive even in the presence of the antibiotic
ampicillin.
BamHI
This is a restriction enzyme which recognizes the base sequence "GGATCC"
and cuts between the two G's on each strand.
band
In gel electrophoresis, a band represents the dyed or stained molecule
you're interested in. In a sample containing two different sized pieces
of DNA, there will be two bands on the gel in that lane. The smaller piece
of DNA will move more quickly through the gel, so it will be farther from
the well. Each band is composed of hundreds of copies of the DNA molecule
of that length. (One DNA molecule would not be detectable.)
base pairs
The length of a piece of DNA is often referred to in terms of how many
base pairs are present in it. For example, the piece below would be 6
base pairs long (the dotted lines represent the sugar-phosphate backbones
of the DNA).
------------ A T C G C A | | | | | | T A G C G T ------------
colony
A colony of is a group of cells which usually form a small white-to-clear
(if E. coli) bump on a plate. Each colony represents millions of
cells, all of which are clones of one original cell.
E. coli
Escherichia coli are bacteria commonly used in research. The strain
we use is not the same as the strain which causes disease.
fragment
A fragment of DNA is a piece of DNA, usually a shorter piece than you
started out with. For example, if you have a circular plasmid of DNA and
you cut it in two places, you will have two fragments of DNA.
gel
electrophoresis
This is a technique used to separate molecules based on size and/or charge.
To separate molecules of DNA, we rely on the negative charge of DNA to
pull it toward the positive electrode. The agarose gel allows smaller
(shorter) lengths of DNA to travel more quickly than longer pieces of
DNA. See the lab manual for details.
HindIII
This is a restriction enzyme which recognizes the base sequence "AAGCTT"
and cuts between the two A's on each strand.
kanamycin
This is an antibiotic which normally can kill E. coli cells.
kanamycin resistance gene
This gene is found in the pKAN plasmid; the protein which this gene codes
for allows an E. coli cell to survive even in the presence of the antibiotic
kanamycin.
lane
In gel electrophoresis, a lane is the section of a gel below one well.
The gels we are using have six wells, so they have six corresponding lanes.
When you look at a stained gel, all the bands in one lane came from the
same sample of DNA loaded into one well.
ligase
This is an enzyme (note the "-ase" ending) which can connect
a DNA backbone. We rely on the sticky ends left from restriction enzyme
cuts to line two pieces of DNA up, but the ligase is necessary to form
the covalent bonds which form the sugar-phosphate backbone.
origin
This is a short sequence of DNA which is necessary for the plasmid to
be replicated within a bacterial cell; without an origin, a plasmid will
not be passed along to a cell's progeny.
pAMP
This is the name given to the plasmid we're using which contains a gene
for resistance to the antibiotic ampicillin. The "p" in pAMP
stands for plasmid. This plasmid has an origin, one BamHI cutting site,
and one HindIII cutting site. (See the pAMP sequence
for more details.)
pKAN
This is the name given to the plasmid we're using which contains a gene
for resistance to the antibiotic kanamycin. The "p" in pKAN
stands for plasmid. This plasmid has an origin, one BamHI cutting site,
and one HindIII cutting site. (See the pKAN sequence
for more details.)
plasmid
This is a small, circular piece of DNA, usually a few thousand base
pairs long, which can be found in bacteria. It is distinct from the bacterial
chromosome. Some types of plasmids are present in large numbers in the
cytoplasm of a bacterial cell, and other types of plasmids are found in
much smaller numbers. A plasmid must contain a sequence called the origin
to replicate; otherwise it will not be maintained in a cell line. Plasmids
represent an incredibly powerful tool for manipulating DNA; they are commonly
used by geneticists for a variety of purposes.
plating
One place to grow cells is on agar plates. The process of spreading cells
onto a plate is called plating. When you plate cells, you can isolate
individual strains of bacteria from one another. Each colony on a plate
consists of millions of cells which arose from a single cell, and are
all identical to that original cell. Not all the colonies on a plate are
necessarily identical.
recombinant
DNA
This is DNA that is newly made, or not in it's orginal form. The term
refers to the fact that you have taken a sequence of DNA and combined
it with another sequence in a new way--you've "recombined" it.
In this lab, we start out with two separate plasmids; when we cut them
apart, we have pieces of our original plasmids. When we ligate these pieces
together, we form brand new combinations of DNA--we make recombinant DNA.
restriction
enzymes
These are enzymes (endonucleases, more specifically) which recognize a
specific, short sequence of DNA and cut the DNA at that point. Different
restriction enzymes recognize and cut different sequences. There are hundreds
of different restriction enzymes available commercially. Many restriction
enzymes leave "sticky ends" when they cut, which are available
to bind with other "sticky ends" left by the same enzymer. Restriction
enzymes are a vital tool in genetics, since they allow cutting (and pasting)
of DNA.
running a gel
Gel electrophoresis (see above) is the process of separating molecules
based on their size and charge. Because the particles move through the
gel during the application of a current, this process is often referred
to as "running a gel."
selection
In our case, we are using the characteristics of the recombinant plasmid
(resistance to kanamycin and ampicillin) to find only those bacteria which
contain that type of plasmid. By plating cells on media containing both
antibiotics, we are selecting cells which meet these criteria.
This type of selection is an artificial selection, imposed by the plates
we make. Artificial selection is used by plant and animal breeders who
want to develop strains of organisms with certain traits. Selection is
a common tool of geneticists.
sticky ends
When certain restriction enzymes cut DNA, they don't cut through both
strands of DNA at the same exact point. We refer to the end they leave
as a "sticky end." One strand is left with unpaired bases, which
can later pair up with another sticky end created by the same enzyme.
Because different restriction enzymes recognize different sequences of
DNA, they leave different unpaired bases, which is why a BamHI sticky
end can't base pair with a HindIII sticky end. (Other restriction enzymes
leave blunt ends (not sticky)--these are not as commonly used by geneticists
because they can only "cut" the DNA, and not "paste"
it.)
transformation
This term refers to the process we use to introduce plasmid DNA into bacterial
cells. Before a bacterial cell can be transformed, it must be made competent
(able to take up DNA).
well
In gel electrophoresis, a well is the small, rectangular shaped pit left
in the surface of the gel by the comb. This is where you load the sample
you're running on the gel. The sample is usually weighted down by glycerol,
so it doesn't come out of the well.
