Cloning vectors for expression-PCR products

Cloning vectors for expression-PCR products.

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...

A positive screen for cloning PCR products.

We have developed a positive screen for cloning PCR products based on translational activation of lacZ. A vector with a translationally deficient lacZ alpha gene has been made by deletion of the Shine-Dalgarno sequence and initiation codon. The Shine-Dalgarno sequence and initiation codon are incorporated into one of the PCR primers to allow complementation by the PCR product of the inactive la...

Directional cloning of PCR products.

INTRODUCTION This protocol is for directional blunt-end cloning of DNA fragments. The target DNA is PCR-amplified, 3'-extensions are polished with Pfu DNA polymerase, and the amplicon is ligated to a blunt-ended plasmid DNA. The products of the ligation reaction are used to transform competent Escherichia coli. A restriction enzyme is added to the ligation reaction (Liu and Schwartz 1992) to re...

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 ...