A novel α-amylase and its application
A technology of amylase and recombinant plasmid, applied in the direction of application, enzyme, introduction of foreign genetic material using vector, etc., to achieve the effect of wide temperature tolerance range
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Embodiment 1
[0012] Example 1: Acquisition of α-amylase gene laxh3357
[0013] Through the analysis of the whole genome of the new deep-sea bacteria XH031, three amylase genes and their gene sequences were obtained, and the upstream primer (5'-CGGAATTCATGCACCCTCGACCGGC-3') and the downstream primer (5'-CCCTCGAGACGCCGCCACATCCG -3') Using genomic DNA as a template, carry out PCR reaction, the PCR reaction composition is as follows (50 μ l reaction system): ddH 2 O 11.5 μl, upstream and downstream primers 0.5 μl each, DNA template 2 μl, 2×GCBuffer 25 μl, Taq 0.5 μl, dNTP Mixture 8 μl. The reaction conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 3 min for 30 s, and final extension at 72°C for 10 min, a total of 30 cycles. After the reaction, the PCR product was recovered to obtain the α-amylase gene laxh3357. laxh3357 is one of the α-amylase genes, its nucleotide sequence is SEQ ID NO: 2, the encoded amino ...
Embodiment 2
[0014] Example 2: Construction of Escherichia coli cloning vector PUCm-T-laxh3357.
[0015] The α-amylase gene laxh3357 was connected to the carrier PUCm-T by using DNA Ligation Kit. The connection system (10 μl) was as follows: SolutionI 5 μl, DNA fragment 4 μl, PUCm-T carrier 1 μl. The connection solution obtained by ligation at 16°C for 16 hours can be used to obtain the E. coli cloning vector PUCm-T-laxh3357, which is used to transform E. coli JM109.
Embodiment 3
[0016] Example 3: Construction of Escherichia coli recombinant strain JM109-PUCm-T-laxh3357
[0017] Add 200 μl of thawed competent Ecoli.JM109 and 10 μl of the connection solution obtained in Example 2 to a 2 ml Eppendorf tube, ice-bath for 30 minutes, heat shock at 42°C for 90 seconds, ice-bath for 15 minutes, add 800 μl of LB medium, and culture with shaking at 37°C for 45 minutes. The bacterial solution was mixed with 4 μl IPTG and 40 μl X-gal, spread on the LB plate containing 100 μg / ml ampicillin, and incubated at 37°C for 12-14h. Pick white colonies for PCR and double enzyme digestion detection, the enzyme digestion system (20μl) is as follows: ddH 2 O 8 μl, PUCm-T-laxh3357 plasmid DNA 8 μl, EcoRI 1 μl, XholI 1 μl, 10×H Buffer 2 μl, those with 1428 bp specific band in agarose gel electrophoresis were positive transformation clones, that is, Escherichia coli JM109- containing PUCm-T-laxh3357 PUCm-T-laxh3357. Send 1ml of the positive clone bacteria solution for testing,...
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