Controls in Molecular Biology



Goals:
  1. Understand the concept of both a positive and negative control
  2. Know the correct controls to use for these Molecular Biology procedures:


Experimental Controls

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.


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:


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:
 

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.
 
 

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:
 



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