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Cloning Vectors and Kits

The cloning of a specific fragment of DNA into a vector such as a plasmid is one of the foundational steps that enables additional characterization of that sequence. Vectors such as the pGEM® series contain multiple cloning sites to excerpt fragments generated by digestion with specific restriction enzymes. Sequences created by PCR and related TA cloning methods can be easily ligated into the pGEM® T-vectors. For multiple downstream applications (i.e., protein expression) the unique Flexi® Cloning System can conveniently transfer fragments to multiple vectors.

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What is a Cloning Vector?

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.