Controls in Molecular Biology
Understand the concept of both a positive
and negative control
Know the correct controls to use for
these Molecular Biology procedures:
restriction digests of starting DNA
transformation of the potential recombinant
detection of positive clones
Controls are important in all
biological disciplines but they are a necessity in Molecular Biology. The
Molecular Biologist can rarely directly observe the processes being studied.
We depend on the controls to assure us that our experimental results are
valid. Each procedure will have its own type of controls, but each should
include an appropriate positive and a negative control.
For each experiment, you should carefully consider
the purpose of your experiment and how you are planning to accomplish this
positive control: a positive control should give the desired outcome
of the experiment, provided that all the reagents and equipment are functioning
properly. For example, if your experiment results in the ability of bacteria
to grow on a petri plate containing antibiotic, your positive control will
be bacteria that are known to carry the appropriate drug resistance marker.
Even if none of your experimental bacteria grow, as long as there is growth
of the positive control you know that growth was posible.
negative control: a negative control should be designed to not
give the desired outcome of the experiment. In the example above, bacteria
which do not carry a drug resistance marker should not be able to grow
on a petri plate containing antibiotic. If growth is observed, it is a
red flag that something is wrong with the experiment. (What could be one
reason for growth?)
There are many steps in producing a
new recombinant plasmid, and each step has its own controls.
A) restriction digests of starting
For example, you may want to
clone a piece of foreign DNA (the insert) into a plasmid (the vector).
The first step is cutting both the insert and the vector with restriction
enzymes. It is important to start with complete digests when cloning. Gel
electrophoresis is used to verify that the DNA has been cleaved completely.
Samples of the plasmid and insert DNA are run on the gel along with:
Uncut plasmid - this is an important control. Uncut plasmid should always
be run with the plasmid digest. By comparing the conformations of uncut
plasmid with the cut plasmid, you can verify that the digest is complete.
By examining the lane of the uncut plasmid, you can judge the purity and
the condition of the DNA.
Uncut insert DNA - whether the fragment is a PCR product or a piece of
another plasmid, you want to be able to compare the size of the cut and
uncut insert DNA. Again, this helps to verify that the digest is complete.
Marker or ladder DNA - a digest containing fragments of known sizes is
used as a giude to confirm that the cut DNA is the expected size. A commonly-used
marker lane is the HindIII digest of Lambda.
B) Transformation of the potential
The process of inserting plasmid
DNA into bacteria (transformation) contains many steps. Cells must be treated
to make them competent to take up plasmid DNA. The cells undergo heat shock
to drive the plasmid into the cell. Cells recover and are finally plated
on antibiotic plates. Thus there are many things that could prevent the
experiment from being successful. The following controls help to monitor
the progress of the experiment:
competent cells without plasmid added - this viability control insures
that the cells have not been killed by the manipulations. This control
uses a sample of the same competent cells used for the rest of the experiment.
The cells are treated the same as the experimental cells through the heat
shock, recovery, and plating steps. However, they do not have a
plasmid added to them. These cells should grow on a plate without antibiotic
(positive control) but not on an antibiotic-containing petri plate (negative
control). The viability control is important when using competent cells
which have been frozen at -80'C and thawed for the experiment.
These controls not only test the
ability of the bacteria to be transformend, they also verify that
the antibiotic resistance plates are functioning properly. Plates with
weak or inactive antibiotic will allow growth of bacterial colonies even
though the colonies do not contain a drug-resistance plasmid.
competent cells transformed with a known plasmid - the competency
control shows that cells are able to take up plasmid DNA. An intact plasmid
is used for transformation. Often, a sample of the intact, uncut vector
plasmid is used. After recovery, the cells are plated on antibiotic-containing
plates. If the cells were indeed competent and the transformation was successful.
then colonies will grow on the petri plate.
C) Detection of positive clones
While other methods are used,
researchers often screen for potential recombinants by performing a small-scale
isolation of plasmid DNA (miniprep) from several colonies. The DNA is cut
with restriction enzymes and analyzed by gel electrophoresis. To verify
that the cloning was successful, you should have DNA bands on the gel that
represent the correct size of both the vector and the insert DNA. The new
construction should have both of these pieces and the size of each can
be matched with the known bands:
cut and uncut vector - the cut vector will show the correct size of the
"backbone" of the new construction. Uncut vector is compared to the cut
vector to verify a complete digest. The uncut vector is also compared to
the uncut plasmid DNA of the potential recombinants. The recombinant plasmids
should be larger than the starting vector.
cut and uncut insert - the cut insert DNA will show the correct size of
the new DNA fragment in the plasmid "backbone".
marker or ladder lane, such as a 1-kb ladder or the HindIII digest of Lambda.
uncut DNA from the experimental samples - this is an important control.
If the new construction is larger than the starting vector (which is often
the case), then the band representing supercoiled DNA should be slightly
closer to the well. Occasionally, the new construction will be present
totally in the DIMER form. If you do not run uncut plasmid but only run
your digest DNA, you will not know this. It can be a problem for you in
later manipulations if you use a dimer plasmid.
Return to Virtual Lab Book Home
Return to Molecular Biology Lab