Web resources for the pUC19 cloning vector plasmid

Manufacturer's information on the pUC19 plasmid

Location of restriction sites on pUC19

pUC19 plasmid nucleotide sequence report from GenBank

 

Background Information

Our ultimate goal over the next few weeks is to show you how DNA is cloned and manipulated in the laboratory, and to show you how molecular techniques can be used to address basic questions in the biological sciences.

In many instances, recombinant DNA techniques are called "cloning" or "gene cloning", but this does not refer to the technology used to produce the famous sheep, Dolly.  "Cloning", in our usage, refers to the isolation some specific piece of DNA, physical attachment to a "vector" for transfer to bacteria for production of many identical copies of this piece of DNA.

Some Basic "Tools" of Recombinant DNA
There are a number of websites that discuss different aspects of recombinant DNA technology. Below are a few general sites that may be of interest:

• Access Excellence Project-Graphics Gallery

You should also check out the Roche Genetics tutorial which can be found on the desktop of the lab computers.

Review of gel electrophoresis
The electrophoretic migration rate of DNA through agarose gels is dependent upon several parameters:

• The molecular size of the DNA. Molecules of linear, duplex DNA (which are believed to migrate in an end-on position) travel through gel matrices at rates that are inversely proportional to the log10 of their molecular weights. Although complicated physically, it is best to think of the separation being due to a molecular sieving or gel filtration effect due to the pore size of the gel matrix. Since the charge to mass ratio is the same independent of the length of the DNA fragment, the separation is not due to differences in charge density as might be expected.

There is a linear relationship between the logarithm of the electrophoretic mobility of DNA (m) and gel concentration, which is described by the equation: logm = logmo - Kr, where mo is the free electrophoretic mobility and Kr is the retardation coefficient, a constant that is related to the properties of the gel and the size and shape of the migrating molecules. Thus, by using gels of different concentrations, it is possible to resolve a wide size-range of DNA fragments. The log10 of the molecular weight (in base pairs) of DNA fragments of known size are plotted versus the distance migrated (in cm).

The agarose concentration. A DNA fragment of a given size migrates at different rates through gels containing different concentrations of agarose.

• The shape of the DNA molecule. Remember that plasmids are circular DNA molecules and can exist as relaxed molecules or they can be supercoiled. When cut with a restriction enzyme, the circular plasmid becomes a linear DNA molecule. Linear DNA molecules run faster than supercoiled circles which run faster than relaxed circular molecules.

Recombinant DNA Tutorials (Cornell Univ.)