PCR Cloning

T-vector cloning, or TA cloning, is a convenient method for cloning PCR products generated with Taq DNA Polymerase. The pGEM®-T vectors are a popular choice for general PCR cloning. These vectors are ready to use in ligation reactions; prepared by cutting with a restriction endonuclease that creates a blunt end and adding a 3´ terminal thymidine (T) to both ends. The 3´ T-overhangs at the insertion site greatly improve ligation efficiency of a PCR product into the plasmid by preventing recircularization of the vector and providing a compatible overhang for PCR products with 5' A-overhangs. The pGEM®-T Easy Vector systems come with competent cells included.

The pTargeT™ Vector is used for cloning and expression of PCR products in mammalian cells.

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An Introduction to PCR Cloning

PCR products generated using a non-proofreading DNA polymerase which lacks 3’ to 5’ exonuclease activity, such as Taq DNA polymerase, are left with a single template-independent nucleotide, deoxyadenosine (A), at the 3´ end of the amplified fragments. This single-nucleotide overhang allows hybridization with and cloning into vectors that have a complementary 3´ single deoxythymidine (T) overhang.

T vectors are linearized plasmids that have been treated to add T overhangs to match the A overhangs of the PCR product. PCR fragments that contain an A overhang can be directly ligated to these T-tailed plasmid vectors with no need for further enzymatic treatment other than the action of T4 DNA ligase.

The high-copy-number pGEM®-T and pGEM®-T Easy Vectors contain T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the coding region for the a-peptide of ß-galactosidase, allowing blue/white selection of recombinant clones.

Both the pGEM®-T and pGEM®-T Easy Vectors contain numerous restriction sites within the multiple cloning region. The pGEM®-T Easy Vector multiple cloning region is flanked by recognition sites for the restriction enzymes EcoRI, BstZI and NotI, providing three single-enzyme digestions for release of the insert. The pGEM®-T Vector cloning region is flanked by recognition sites for the enzyme BstZI. Alternatively, a double digestion may be used to release the insert from either vector.