Directional cDNA library construction assisted by the in vitro recombination reaction

We report here a new directional cDNA library construction method using an in vitro site-specific recombination reaction, based on the integrase–excisionase system of bacteriophage λ. Preliminary experiments revealed that in vitro recombinational cloning (RC) provided important advantages over conventional ligation-assisted cloning: it eliminated restriction digestion for directional cloning, generated low levels of chimeric clones, reduced size bias and, in our hands, gave a higher cloning efficiency than conventional ligation reactions. In a cDNA cloning experiment using an in vitro synthesized long poly(A)+ RNA (7.8 kb), the RC gave a higher full-length cDNA clone content and about 10 times more transformants than conventional ligation-assisted cloning. Furthermore, characterization of rat brain cDNA clones yielded by the RC method showed that the frequency of cDNA clones >2 kb having internal NotI sites was ∼6%, whereas these cDNAs could not be cloned at all or could be isolated only in a truncated form by conventional methods. Taken together, these results indicate that the RC method makes it possible to prepare cDNA libraries better representing the entire population of cDNAs, without sacrificing the simplicity of current conventional ligation-assisted methods.

Library construction, architecture, fittings, and furniture /

Includes index.

M.U. Library construction [Front (north) of General Library]

West stack. Albert Kahn, architect. Selden Breck contractors built stacks 1916-1918. Construction of General Library Building authorized June 1916. Two stacks built at right angles to 1898 stacks, which were retained. In 1918 Old Library was demolished and front (north side) of General Library begun. Completed in 1920. Photograph mounted on linen. From construction photo album received by Buildings and Grounds

M.U. Library construction [Front (north) of General Library]

Front (north) of General Library. Albert Kahn, architect. Selden Breck contractors built stacks 1916-1918. Construction of General Library Building authorized June 1916. Two stacks built at right angles to 1898 stacks, which were retained. In 1918 Old Library was demolished and front (north side) of General Library begun. Completed in 1920.

The Olive Tree, Vol. 7 Number 1, 1999

The Summer 1999 issue of The Olive Tree features articles about library projects, collections, technological innovations, and events at Fogler Library, University of Maine.

Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-pax genes

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.

Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 × 109 members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody–antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.

University of Michigan Library. Selden Breck Construction [West Stack]

West stack. Albert Kahn, architect. Selden Breck contractors built stacks 1916-1918. Construction of General Library Building authorized June 1916. Two stacks built at right angles to 1898 stacks, which were retained. In 1918 Old Library was demolished and front (north side) of General Library begun. Completed in 1920. Photograph mounted on linen. From construction photo album received by Buildings and Grounds