Effect of growth temperature and vector ends phosphorylation in the cloning efficiency of specific DNA fragments.

Subcloning of DNA fragments into plasmid vectors is a routine procedure in molecular biology laboratories. However, occasionally DNA fragments are difficult to clone into specific vectors, resulting in low efficiency of isolation of individual constructs. During subcloning experiments for the study of regulation of expression of the mouse gamma-glutamyl transpeptidase gene (GGT) gene, we encountered difficulties in cloning a 416-bp DNA fragment that includes part of the untranslated region of the type II GGT RNA and 346 bp of the type II promoter region (4). In this study, we tested whether omitting the alkaline phosphatase treatment of the cloning vector 5′ ends and lowering the growth temperature of transformed bacteria to 30°C would result in higher yields of plasmid-insert constructs. The GGT type II 416-bp fragment was obtained by polymerase chain reactin (PCR) amplification of a plasmid containing a 6.0-kb insert of the GGT promoter region (3). The amplifying primers contained a XhoI restriction site to be used for subsequent subcloning. The PCR products were subcloned into the pT7Blue vector and transfected into NovaBlue Competent Cells (both from Novagen, Madison, WI, USA). Individual clones of the pT7Blue vector containing the insert were digested with XhoI to release the GGT type II 416-bp fragment, which was then used for subcloning experiments into the promoterless vector pJFCAT (1). The pJFCAT vector was linearized with XhoI, followed by treatment with alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN, USA), at 37°C for 60 min, and inactivation of alkaline phosphatase by heating the reaction at 65°C for 10 min. The ligation of the GGT type II 416-bp XhoI fragment into pJFCAT was performed using equimolar amounts of vector and insert; it was ligated overnight at 16°C, with T4 DNA Ligase (Promega, Madison, WI, USA) in a 10-μL final volume. The ligation products (1 μL) were used to chemically transform NovaBlue Competent Cells, as described by the manufacturer. Because this procedure did not yield positive clones containing the insert, and the overall number of bacterial clones was small, we also transformed Epicurian Coli SURE ElectroporationCompetent Cells (Stratagene, La Jolla, CA, USA). After transformation, bacterial cells were grown for 1 h at 37°C, plated and grown overnight at 37°C onto ampicillin plates. All of the isolated clones invariably yielded a combination of the original vector (pJFCAT), possibly resulting from intramolecular ligation of non-dephosphorylated plasmid and a plasmid of abnormal size (Figure 1); however, no clones containing the GGT type II 416-bp XhoI fragment were identified. The clones were screened by digestion with AatII (Boehringer Mannheim), because one recognition site for this enzyme is present in the cloning vector (pJFCAT), and another site is present within the GGT type II 416-bp XhoI fragment. The expected results after digestion of pJFCAT clones containing the GGT type II 416-bp XhoI fragment with AatII include: (i) 5.6-kb plasmid, representing linear forms of the pJFCAT cloning vector, and (ii) clones containing the insert should release a 1.4-kb fragment after digestion with the enzyme (Figure 1). However, we could only identify linear forms of pJFCAT and plasmid forms with abnormal molecular weight. These results surprised us because we have previously cloned larger fragments of the GGT type II promoter region containing the 416 bp into the pJFCAT vector (3). A previous report indicated that growing bacterial clones at 30°C, after transformation of bacteria with ligation products resulted in a higher level of recovery of inserts (2). Therefore we tried to grow the bacteria after transformation with the ligation products at either 37° or 30°C. In addition, we used pJFCAT with and without alkaline phosphatase treatment in the ligation reactions. The cloning vector (pJFCAT) was linearized with XhoI, either treated with alkaline phosphatase or not and was then isolated by gel purification to decrease the possibility of contamination with non-linearized vector. The GGT type II 416-bp region was released from pT7Blue after digestion with XhoI, and then the fragment was isolated by gel purification. Approximately equimolar amounts of vector and insert were ligated overnight at 16°C. Electroporationcompetent SURE cells were transformed and, after shocking, were allowed to grow for 1h at either 37° or 30°C and then plated onto ampicillin plates at the same temperatures. Twenty Benchmarks