Nucleotide specific to ITS of Balcillus proteus mirabilis and use thereof
A technology of Proteus mirabilis and nucleotides, which is applied in the field of nucleotides, can solve the problems of complex detection operation process, high technology and equipment conditions, long detection cycle, etc., and achieve simple test equipment, simple preparation method and long detection cycle. short effect
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Embodiment 1
[0084] Example 1 : Genome Extraction
[0085] Proteus mirabilis 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. Genomic ...
Embodiment 2
[0086] Example 2 : Amplification of ITS in Proteus mirabilis by PCR
[0087] The ITS of Proteus mirabilis was amplified by PCR using the genome of Proteus mirabilis as a template. First, design an upstream primer (5'-TGTACA CAC CGCCCG TC-3') based on the conserved region of the 16S rRNA gene at one end of the ITS, and then design a downstream primer (5'-GGTACT TAG ATG TTT CAG TTC-3 '). The PCR reaction program is as follows: pre-denaturation at 94°C for 5 minutes; then denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 1 minute, thus performing 30 cycles; finally, extending at 72°C for 5 minutes to obtain PCR The size and specificity of PCR products were detected by 0.8% agarose gel electrophoresis. Combine 3 tubes of long PCR products, and recover the shortest band in the purified PCR products with UNIQ-10 Column DNA Gel Recovery Kit from Shanghai Sangon Bioengineering Technology Company;
Embodiment 3
[0088] Example 3 : build its clone
[0089] The first is the acquisition of the connection product:
[0090] The PCR purified product was mixed with Promega's 3×10 -3 The pGEM-T-Easy vector was ligated at 16°C for 24 hours, with a total volume of 10 μl, which contained 1 μl of 10×buffer and 0.5 units of T4 DNA ligase to obtain the ligation product.
[0091] Followed by the preparation of competent cells:
[0092]Competent cells Escherichia coli DH5α were prepared according to the method provided by Bio-Rad. Take a single colony of Escherichia coli DH5α in 5ml of LB medium, culture it at 180rpm for 10 hours, take 2ml of the culture and transfer it to 200ml of LB medium, shake vigorously at 37°C and 250rpm to about OD6000.5, then freeze Bath cooled for 20 minutes and centrifuged at 4000 rpm for 15 minutes at 4°C. Drain the supernatant, blow off the bacteria with 200ml of cold ice-precooled deionized sterilized water, and centrifuge at 4000rpm at 4°C for 15 minutes. Blow o...
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