Sereening of transfected bacteria by bacteria colonies PCR and plas-mid PCR

Abstract：Objective We have developed a procedure that allows rapid screening of transfected bacteria colonies and di-rect sequencing of bacteria colonies as well as plasmid PCR-products．Methods In the first step the multiple cloning sites con-taining the sequences of interest are amplified by bacteria colonies PCR and plasmid PCR using lacZ specific primers in cloned vectors．For screening of expression vectors of VH and VL of monoclonal antibodies we have used family specific primers．Colonies PCR-and plasmid PCR products are purified enzymaticaly and are used as templates in dye-primer or dye-terminator sequencing.Results The procedure allows fast sequencing of a large numbers of samples with minimal on-hands time and minimal need for precipitation/centrifugation steps．Using this method，we routinely can read 600 or more nucleotides for 120 PCR-products on a standard ABI 373A sequencer．Conclusions Despite of the fact that our methods requires an additional PCR-step,its faster，cheaper and more reliable than the standard method using highly puuified plasmid DNA．