An Efficient Method to Prepare PCR Cloning Vectors

An Efficient Method to Prepare PCR Cloning Vectors

An improved procedure for preparing PCR cloning vectors was developed. This procedure includes the incorporation of adapters to create XcmI restriction enzyme sites in pBluescript II SK(+) vectors, digestion with XcmI followed by further digestion of the small fragment produced by XcmI digestion with additional enzymes, and purification with PCR purification kits. Using this procedure, PCR clon...

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 new method to customize protein expression vectors for fast, efficient and background free parallel cloning

BACKGROUND Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any...

Reliable PCR-based method for cloning cDNA in plasmid vectors by frequency estimation.

products by generating sticky ends for ligation. This step is not essential and should be omitted when using a bluntcutting restriction endonuclease that is inhibited by methylation. The results from applying our approach demonstrate the mutation of three bases of a HBV sequence using an alternative PCR-based, SDM method. The technique obviates the variable efficiency of using doublestranded me...