Magnetic bead capture of cDNAs from double-stranded plasmid cDNA libraries.
We have developed a cDNA library screening method which allows the simultaneous screening of >10 ( 12 ) double-stranded plasmid cDNA molecules with minimal a priori sequence knowledge. A biotinylated, gene-specific oligonucleotide probe along with abutting 'blocking' oligos is hybridized to the plasmid cDNA library and the target plasmid retrieved with paramagnetic streptavidin beads and transformed into Escherichia coli. Multiple rounds of enrichment with a target plasmid represented at 0.002-0.0001% resulted in over one-third positive clones. Our method will be useful for isolating even the rarest cDNAs starting from ESTs, isolated exons or homologous sequence information.
Shepard
A R
AR
Departments of Physiology/Biophysics and Ophthalmology, Mayo Foundation, 200 1st Street SW, Rochester, MN 55905, USA.
Rae
J L
JL
D001426:Bacterial Proteins; D018076:DNA, Complementary / Q000302:isolation & purification*; D015723:Gene Library; D008863:Microspheres; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D019809:Streptavidin
D001426:Bacterial Proteins; D018076:DNA, Complementary; D019809:Streptavidin