菌落PCR和质粒PCR对转化菌的筛选

Screening of transfected bacteria by bacteria colonies PCR and plasmid PCR

目的为建立简便、可靠转化菌的筛选方法。方法应用菌落 PCR和重组质粒 PCR方法对转化菌进行筛选和DNA序列分析。克隆载体和表达载体筛选及鉴定采用与载体多克隆插入位点序列互补的通用引物或免疫球蛋白家族特异性引物。阳性菌落和质粒 PCR产物通过酶消化后作为模板进行自动测序。结果菌落 PCR和质粒 PCR技术可作为基因克隆筛选和鉴定的方法。结论菌落 PCR和质粒 PCR方法简便、快速、可靠 ,其产物可作为模板直接用于序列分析。

ObjectiveWe have developed a procedure that allows rapid screening of transfected bacteria colonies and direct sequencing of bacteria colonies as well as plasmid PCR products.MethodsIn the first step the multiple cloning sites containing 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.ConclusionsDespite of the fact that our methods requires an additional PCR step,its faster,cheaper and more reliable than the standard method using highly purified plasmid DNA. [