Nucleotide peculiar to 0-antigen of 12 type Baoshi Sh.dysenterae
A Shigella baumannii, nucleotide technology, applied in sugar derivatives, biochemical equipment and methods, applications, etc., can solve problems such as false positives
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Embodiment 1
[0054] Example 1: Genome extraction.
[0055] Shigella baumannii type 12 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 then extract twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), take the supernatant, and then extract with an equal volume of ether. Extract 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. Gen...
Embodiment 2
[0056] Example 2: Amplification of the O-antigen gene cluster in Shigella baumannii type 12 by PCR
[0057] Using the genome of Shigella baumannii type 12 as a template, its O-antigen gene cluster was amplified by Long PCR. First, the upstream primers (5'-ATT GTG GCT GCA GGG ATC AAA GAA ATC-3') were designed according to the galF gene often found upstream of the O-antigen gene cluster, and then the downstream primers were designed according to the gnd gene downstream of the O-antigen gene cluster ( 5'-TAG TCG CGC TGN GCC TGG ATT AAG TTC GC-3'). The O-antigen gene cluster was amplified using the Expand Long Template PCR method of Boehringer Mannheim. The PCR reaction procedure was as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, annealing at 61°C for 30 seconds, and extension at 68°C for 15 minutes. Carry out 30 cycles in this way; finally, continue to extend at 68° C. for 7 minutes to obtain PCR products, and use 0.8% agarose gel e...
Embodiment 3
[0058] Example 3: Construction of O-antigen gene cluster library.
[0059] 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, once with a mixed solution of phenol:chloroform:isoamyl alcohol (25:24:1), and then extract with an equal volume of ether After one time, the DNA was precipitated with 2.5 times the volume of absolute ethanol, washed with 70% ethanol, and finally resuspended in 18ul of water. Then add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25ul 100mM DTT and 5 units of T4 DNA polymer...
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