45 nucleotides of homology are added directly to each primer, the lengths of the HRs in the short-primer method can be very large (e.g. several hundred bp) to increase the recovery of recombinants. The length of a homology
region is limited only by the conditions of the PCR. To demonstrate the efficacy of the short-primer Selleck Ku 0059436 method, we compared the frequency of recombinants obtained with the long-primer method (50 nucleotides of homology on each primer) to that obtained by the short-primer method (HRI = 200 bp; HRII = 250 bp). In both, the lacZα-MCS::aacC1 replaced the MCS of pJAK12 (see Fig. 2b). About 200 Gmr transformants mL−1 were obtained with the long-primer method, whereas the short-primer method gave over 4000 Gmr transformants mL−1 with equal amounts of DNA. The results indicated not only that recombinants were obtained with the short-primer method but also that the larger HRs in the method make it easier to obtain the desired recombinant. We wholeheartedly thank Dr Robert Washburn for his advice on recombineering and
for the gift of strain RSW358. We are grateful to Dr Donald Court for generously providing the pSIM9 plasmid and its sequence and to Dr Michael Kovach for plasmid pBBR1MCS. This work was funded by National Institutes of Health grant R01-DE14713 to D.H.F. K.J.R. was partially supported by the Columbia University Work Exemption Program. “
“Nosemosis is a contagious disease of honeybees (Apis mellifera) manifested by increased buy Tamoxifen winter mortality, poor spring build-up and even the total extinction of infected bee colonies. In this paper, loop-mediated isothermal amplifications (LAMP) were used for the first time to identify and differentiate N. apis and N. ceranae, the causative agents of nosemosis. LAMP assays were performed
at a constant temperature of 60 °C using two sets of six species-specific GNAT2 primers, recognising eight distinct fragments of 16S rDNA gene and GspSSD polymerase with strand displacement activity. The optimal time for LAMP and its Nosema species sensitivity and specificity were assessed. LAMP only required 30 min for robust identification of the amplicons. Ten-fold serial dilutions of total DNA isolated from bees infected with microsporidia were used to determine the detection limit of N. apis and N. ceranae DNAs by LAMP and standard PCR assays. LAMP appeared to be 103-fold more sensitive than a standard PCR in detecting N. apis and N. ceranae. LAMP methods developed by us are highly Nosema species specific and allow to identify and differentiate N. apis and N. ceranae. “
“Listeria monocytogenes (LM) is a zoonotic pathogen that widely adapts to various environments. Recent studies have found that noncoding RNAs (ncRNAs) play regulatory roles in LM responses to environmental stress.