Klonierungssysteme und Kompetente Zellen

There are many types of cloning vector, but the most commonly used are plasmids. A basic plasmid vector contains features that allow the convenient insertion of a DNA fragment into the vector for subsequent manipulation or analysis. In classic subcloning, the vector and sequence of interest are cut with a specific restriction enzyme and the resulting digested plasmid and DNA sequence of interest are joined together by molecular ligation using T4 DNA Ligase.

TA cloning is a technique that avoids the use of restriction enzymes and is thus easier and faster than traditional subcloning. The technique relies on the ability of adenine (A) and thymine (T) on different DNA fragments to hybridize and, in the presence of T4 DNA ligase, become ligated together. PCR products are usually amplified using Taq DNA polymerase, which preferentially adds an adenine to the 3' end of the amplification product. In TA cloning, such PCR amplified inserts are cloned into linearized “T”-vectors that have complementary 3' thymine overhangs.