Nucleotide and its use
A technology of Shigella baumannii and nucleotides, which can be used in the fields of application, microbiological determination/testing, sugar derivatives, etc., and can solve the problems of indistinguishability
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Embodiment 1
[0045] Shigella baumannii type 13 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), and extract the supernatant with an equal volume of ether to remove residual phenol. The supernatant was precipitated with 2 times the volume of ethanol, the DNA was rolled out with glass wool and washed with 70% ethanol, the DNA was resuspended in 30ul TE, and the genomic DNA was detected by 0.4% agarose gel electrophoresis. Example 2: Amplificati...
Embodiment 2
[0046] The O-antigen gene cluster of Shigella baumannii type 13 was amplified by Long PCR. First design the upstream primer (5'-ATT GTGGCT GCA GGG ATC AAA GAA AT-3') based on the JumpStart sequence often found in the promoter region of the O-antigen gene cluster, and then design the downstream primers based on the gnd gene downstream of the O-antigen gene cluster (5'-TAG TCG CGT GNG CCT GGA TTA AGT TCG C-3'). Use the Expand Long Template PCR method of BoehringerMannheim to amplify the O-antigen gene cluster. The PCR reaction program is as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 68°C for 15 minutes. Do 30 cycles. 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 p...
Embodiment 3
[0046] The O-antigen gene cluster of Shigella baumannii type 13 was amplified by Long PCR. First design the upstream primer (5'-ATT GTGGCT GCA GGG ATC AAA GAA AT-3') based on the JumpStart sequence often found in the promoter region of the O-antigen gene cluster, and then design the downstream primers based on the gnd gene downstream of the O-antigen gene cluster (5'-TAG TCG CGT GNG CCT GGA TTA AGT TCG C-3'). Use the Expand Long Template PCR method of BoehringerMannheim to amplify the O-antigen gene cluster. The PCR reaction program is as follows: pre-denaturation at 94°C for 2 minutes; then denaturation at 94°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 68°C for 15 minutes. Do 30 cycles. 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 p...
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