Nucleotide specific against 0-antigen of colibacillus 0107
A technology of Escherichia coli and nucleotides, which is applied in the determination/testing of microorganisms, sugar derivatives, biochemical equipment and methods, etc., and can solve problems such as false positives
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Embodiment 1
[0045] Escherichia coli O107 was cultured overnight at 37°C in 5 mL of LB medium, and the cells were collected by centrifugation. The cells were resuspended with 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, incubated at 37°C for 20 minutes, and then 10ul 10mg / ml lysozyme was added to continue the incubation for 20 minutes. Then add 3ul 20mg / ml proteinase K, 15ul 10% SDS, incubate at 50°C for 2 hours, then add 3ul 10mg / ml RNase, and incubate at 65°C for 30 minutes. Add an equal volume of phenol to extract the mixture, take the supernatant and extract twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) mixed solution, take the supernatant and extract with an equal volume of ether to remove residual phenol. The DNA was precipitated with 2 times the volume of ethanol in the supernatant, and the DNA was rolled out with glass wool and washed with 70% ethanol, and finally the DNA was resuspended in 30ul TE. Genomic DNA was detected by 0.4% agarose gel elect...
Embodiment 2
[0046] Using the genome of Escherichia coli O107 as a template, its O-antigen gene cluster was amplified by Long PCR. First, design the upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) based on the JumpStart sequence often found in the promoter region of the O-antigen gene cluster, and then design the downstream primer (# 1524-TAG TCG CGT GNG CCTGGA TTA AGT TCG C); the O-antigen gene cluster was amplified by the Expand Long Template PCR method of Boehringer Mannheim, and the PCR reaction procedure was as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, 30 cycles of annealing at 60°C for 30 seconds and extension at 68°C for 15 minutes. Finally, continue extending at 68° C. for 7 minutes to obtain PCR products, and use 0.8% agarose gel electrophoresis to detect the size and specificity of the PCR products. Combine 6 tubes of long PCR products, and use Promega's Wizard PCR Preps purification kit to purify the PCR products. Emb...
Embodiment 3
[0046] Using the genome of Escherichia coli O107 as a template, its O-antigen gene cluster was amplified by Long PCR. First, design the upstream primer (#1523-ATT GTG GCT GCA GGG ATC AAA GAA AT) based on the JumpStart sequence often found in the promoter region of the O-antigen gene cluster, and then design the downstream primer (# 1524-TAG TCG CGT GNG CCTGGA TTA AGT TCG C); the O-antigen gene cluster was amplified by the Expand Long Template PCR method of Boehringer Mannheim, and the PCR reaction procedure was as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, 30 cycles of annealing at 60°C for 30 seconds and extension at 68°C for 15 minutes. Finally, continue extending at 68° C. for 7 minutes to obtain PCR products, and use 0.8% agarose gel electrophoresis to detect the size and specificity of the PCR products. Combine 6 tubes of long PCR products, and use Promega's Wizard PCR Preps purification kit to purify the PCR products. Emb...
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