a highly efficient cloning vector was constructed for cloning pcr products by inserting an 80 bp dna fragment into pgem-5zf (+) vector. the xcm i digestion of this vector gave rise to a 3’ overhanging deoxythymidine offering the possibility of cloning pcr products with 3' adenosine overhang created by taq dna polymerase. furthermore, two ecor i sites were added to the construct for identificati...

INTRODUCTION This protocol is for directional blunt-end cloning of DNA fragments. The target DNA is PCR-amplified, 3'-extensions are polished with Pfu DNA polymerase, and the amplicon is ligated to a blunt-ended plasmid DNA. The products of the ligation reaction are used to transform competent Escherichia coli. A restriction enzyme is added to the ligation reaction (Liu and Schwartz 1992) to re...

conclusions l. innocua is not a pathogen, but the presence of the bacterium could be an indicator of probable contamination with l. monocytogenes. moreover, there is a potential risk to public health from the consumption of raw or undercooked burgers, which may increase the possibility of the acquisition of resistance to antibiotics. objectives the aim of this study was to evaluate the occurren...

In many DNA amplification reactions, extra fragments arising from nonspecific mispriming, a mixture of target templates or allelic variation may be generated in addition to the desired product. Some nonspecific bands can be reduced by more stringent annealing conditions, but in cases of allelic variation or other mixed targets, the amplification products may be of the same length but differ by ...