Nucleotide with O-antigen idiocrasy against Ball's and shiga's-8 and colibacillus 0143
A technology of Shigella baumannii and Escherichia coli, applied in the field of nucleotides specific to the O-antigen of Shigella baumannii type 8 and Escherichia coli O143, which can solve the problems of indistinguishability
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Embodiment 1
[0045] Example 1: Extraction of Genome
[0046] Shigella 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 of 50mM Tris-HCl (pH8.0) and 10ul of 0.4M EDTA, incubated at 37°C for 20 minutes, and then 10ul of 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, 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), and extract the supernatant with an equal volume of ether to remove residual phenol. The supernatant was used to precipitate DNA with 2 times volume of ethanol, 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 electro...
Embodiment 2
[0047] Example 2: Amplification of the O-antigen gene cluster in Shigella baumannii type 8 by PCR
[0048] The O-antigen gene cluster of Shigella baumannii type 8 was amplified by Long PCR. First, design the upstream primer (#1523-ATTGTG 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 CCT GGA TTA AGT TCG C). Use the Expand Long Template PCR method of BoehringerMannheim to amplify the O-antigen gene cluster. The PCR reaction procedure is as follows: pre-denature at 94°C for 2 minutes; then denature at 94°C for 10 seconds, anneal at 60°C for 30 seconds, and extend at 68°C for 15 minutes. Do 30 cycles. Finally, continue extending at 68° C. for 7 minutes to obtain the PCR product, and use 0.8% agarose gel electrophoresis to detect the size and specificity of the PCR product. Combine 6 tubes of long PCR products, and use Promega's Wizard PCR...
Embodiment 3
[0049] Embodiment 3: construct O-antigen gene cluster library:
[0050] The first is the acquisition of the ligation product: use the modified Novagen DNaseI shot gun method to construct the O-antigen gene cluster library. The reaction system is 300ng PCR purified product, 0.9ul 0.1M MnCl 2 , 1 ul of 1 mg / ml DNaseI diluted 1:2000, and the reaction was carried out at room temperature. Digest for 10 minutes to concentrate the DNA fragment size between 1kb-3kb, then add 2ul 0.1M EDTA to terminate the reaction. Combine 4 tubes of the same reaction system, extract once with an equal volume of phenol and phenol:chloroform:isoamyl alcohol (25:24:1), and then extract twice with an equal volume of diethyl ether, then use 2.5 times the volume of The DNA was precipitated with absolute ethanol, washed with 70% ethanol, and finally resuspended in 18ul of water. Then add 2.5uldNTP (1mMdCTP, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25ul 100mM DTT and 5 units of T4 DNA polymerase to this mixture, 11...
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