36 results about "Escherichia coli DNA" patented technology

A process for preparing high-purity DNA photorepairase includes such steps as sing gentic engineering method to obtain the DNA photorepairase with His marker of colibacillus, and affinity chromatography by Ni-ion affinity chromatographic column. Its advantages are high purity (90%) short period, and low cost.

This invention provides a kind of improved construction method of BAC subclone base. The processing steps are: extraction and purification of BAC DNA, the fragememtation and end trimming of BAC DNA; the recollection of target fragment and the attachment of vector; the transformation of attachment. For example, the BAC subclone base built by corn is with more than 1500 clones. The empty clone and coliform DNA pollution rate is lower than 5%. The inserted fragment is 3kb in average. This method is easy, cheap, fast and effective.

The invention discloses multi-PCR detection primers for detecting four sheep pathogenic bacteria and a detection method. The primers include Klebsiella pneumoniae upstream and downstream primers, proteus mirabilis upstream and downstream primers, escherichia coli upstream and downstream primers and salmonella upstream and downstream primers. According to the detection method, the primers are adopted to perform multi-PCR amplification reaction with Klebsiella pneumoniae DNA, proteus mirabilis DNA, escherichia coli DNA and salmonella DNA as templates, PCR amplification reaction products are added into loading buffer, the mixture is put into agarose gel containing ethidium bromide, and electrophoresis detection is performed to verify the specificity of the primers. The detection method has the advantages that molecular detection with high sensitivity can be achieved just through conventional instruments, and the detection sensitivity of the method is high; meanwhile, no complex operation system is needed, and detection can be performed under common laboratory conditions; besides, the method is easy and fast to implement, high in sensibility and the like.

The invention discloses escherichia coli DNA photolyase and a construction method thereof. The enzyme has the amino acid sequence shown in SEQ ID NO: 1 (in the description). The construction method comprises the following steps: obtaining a DNA photolyase gene from the conventional WT escherichia coli; designing a mutation primer to obtain a mutational gene segment; connecting the mutational gene segment with pET22b plasmid; constructing recombinant expression plasmid pET22b-A377S; converting the recombinant expression plasmid into a competent cell of escherichia coli; constructing genetically engineered bacterium BL21 (DE3) / pET22b-A377S for mutant enzyme expression. According to the invention, better expression is achieved under the conditions of 18 DEG C and 1 mM ITPG; the expressed photolyase is more stable in activity, and the antioxidant effect of the enzyme is remarkably improved.

A method for determining the escherichia coli DNA content of a biological product is provided. The method includes subjecting the biological product to real-time fluorescence quantitative PCR detection by utilizing an escherichia coli specific primer group to determine the escherichia coli DNA content of the biological product. By utilizing the method in an embodiment, the escherichia coli DNA content of the biological product can be quantificationally analyzed, operation is simple and convenient, time consumed is short, sensitivity is high, and the lowest detection limit of the escherichia coli DNA content of the biological product can be 2.0 fg / microlitre. The method can provide reliable quality control bases for production of biotech medicines adopting escherichia coli as a host, and provides important supports for development and safe production of recombinant biotech medicines.