Reliable PCR-Based Method for Cloning cDNA in Plasmid Vectors by Frequency Estimation

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...

Cloning vectors for expression-PCR products.

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 yeast telomeres on linear plasmid vectors.

We have constructed a linear yeast plasmid by joining fragments from the termini of Tetrahymena ribosomal DNA to a yeast vector. Structural features of the terminus region of the Tetrahymena rDNA plasmid maintained in the yeast linear plasmid include a set of specifically placed single-strand interruptions within the cluster of hexanucleotide (C4A2) repeat units. An artificially constructed hai...

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...