Controls in Molecular Biology

1. Understand the concept of both a positive and negative control
2. 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 purpose.

• 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?)

Cloning of DNA

    There are many steps in producing a new recombinant plasmid, and each step has its own controls.
 

A) restriction digests of starting DNA

    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 recombinant

    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.

• 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 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.