construction of a high efficiency pcr products cloning t vector using pgem-5zf (+)

Construction of a High Efficiency PCR Products Cloning T Vector Using pGEM-5zf (+)

A highly efficient cloning vector was constructed for cloning PCR products by inserting an 80 bp DNA fragment into pGEM-5zf (+) vector. The Xcm I digestion of this vector gave rise to a 3' overhanging deoxythymidine offering the possibility of cloning PCR products with 3' adenosine overhang created by Taq DNA polymerase. Furthermore, two EcoR I sites were added to the construct for identificati...

High-efficiency T-vector cloning of PCR products by forced A tagging and post-ligation restriction enzyme digestion.

Cloning PCR products into T vectors.

MATERIALS Bacteriophage T4 DNA ligase T vector Target DNA (25 μg/ml), amplified by PCR When the PCR mixture contains more than one or two bands of amplified DNA, purify the target fragment by electrophoresis through low melting/gelling temperature agarose (please see Recovery of DNA from Low-meltingtemperature Agarose Gels: Organic Extraction). If not purified by gel electrophoresis, PCR-amplif...

Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products.

Although the polymerase chain reaction (1,2) (PCR) can be used to produce a large amount of a specific DNA from a complex source, cloning the PCR products has not proven to be straightforward. Restriction endonuclease sites are often incorporated into the oligonucleotide primers used for amplification, so that cleavage of the product will create sticky ends that can theoretically be ligated to ...

pPCV, a versatile vector for cloning PCR products

The efficiency of PCR product cloning depends on the nature of the DNA polymerase employed because amplicons may have blunt-ends or 3' adenosines overhangs. Therefore, for amplicon cloning, available commercial vectors are either blunt-ended or have a single 3' overhanging thymidine. The aim of this work was to offer in a single vector the ability to clone both types of PCR products. For that p...

Construction of T-vector derived from pBluescript ΙΙ SK with a positive selection marker, a rapid system for cloning

A rapid DNA cloning system is a research interest of many scientists. TA cloning is one of the methods used for the cloning of PCR-amplified DNA molecules. The TA cloning method is a convenient and labor-saving replacement to traditional, restriction enzyme-mediated cloning strategies. A T-vector called pBlueskript ΙΙ SK-1 with the lethal gene ccdB was designed to construct a positive selection...