Normalization of a cDNA Library Cloned in λZAP by a Long PCR and cDNA Reassociation Procedure

Normalization of a cDNA Library Cloned in λZAP by a Long PCR and cDNA Reassociation Procedure

250 BioTechniques Vol. 34, No. 2 (2003) be expected from an amplificationbased strategy. To our knowledge, this is the first time that such analysis has been conducted to assess the representation of a genomic library. In summary, we have developed a quick, facile protocol for constructing libraries from relatively small amounts of DNA. Using our method, we have shown that a representative, ran...

cDNA Microarray Data Normalization

PCR-based cDNA library construction: general cDNA libraries at the level of a few cells.

A procedure for the construction of general cDNA libraries is described which is based on the amplification of total cDNA in vitro. The first cDNA strand is synthesized from total RNA using an oligo(dT)-containing primer. After oligo(dG) tailing the total cDNA is amplified by PCR using two primers complementary to oligo(dA) and oligo(dG) ends of the cDNA. For insertion of the cDNA into a vector...

Systematic screening of an arrayed cDNA library by PCR.

We have developed a PCR-based method for rapid and effective screening of arrayed cDNA libraries. This strategy directly addresses the limitations of conventional hybridization-based schemes and provides a more rapid, cost-effective, and sensitive method compatible with large-scale and routine cDNA clone recovery. To prepare arrayed libraries, 1-2 x 10(6) cDNA clones were propagated as individu...

NORMALIZATION OF cDNA LIBRARIES

In a cellular transcriptome, the number of mRNA copies per gene may differ by several orders. In cDNA libraries, performed from mRNA, these proportions are the same. Normalization methods allow us to equalize numbers of gene’s copies in the library. Normalized cDNA libraries are used to discover new genes transcribed at relatively low levels or for functional screenings. Here, we observed diffe...