Clone of novel beta-mannanase gene and preparation method of recombinase thereof
A mannanase, a new type of technology, applied in genetic engineering, DNA preparation, plant genetic improvement and other directions, can solve problems that have not been reported in research, and achieve the effects of large-scale industrial production, high catalytic activity and thermal stability
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Embodiment 1
[0038] Cloning of the 3' end mRNA sequence of embodiment 1β-mannanase gene
[0039] The first strand of cDNA was synthesized by reverse transcription with Oligo dT-Adaptor Primer as primers; the first round of PCR was performed with M13 Primer M4 and Man5A-3F1 as primers, and the reaction conditions were: 94°C for 2min, 30 cycles (94 ℃ for 30s, 53℃ for 30s, 72℃ for 90s), 72℃ for 10min; use M13 Primer M4 and Man5A-3F2 as primers for the second round of PCR, and the reaction conditions are: 94℃ for 2min, 30 cycles (94℃ for 30s, 53 ℃ 30s, 72℃ 90s), 72℃ 10min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered and ligated with pUCm-T (pUCm-T-man5A3′), transformed into JM109, and sent to Shanghai Sangong for sequencing after enzyme digestion and identification. Aus man5A 3' mRNA sequence.
Embodiment 2
[0040] Cloning of the 5' end mRNA sequence of embodiment 2β-mannanase gene
[0041] The first round of PCR was performed using the Outer Primer and Man5A-5R1 of the 5′-Full RACE Kit as primers. The reaction conditions were: 94°C for 3min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), 72°C 10 min; the second round of PCR was performed using Inner Primer and Man5A-5R2 as primers, and the reaction conditions were: 94°C for 3 min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), and 72°C for 10 min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered by tapping the gel and ligated with pUCm-T (pUCm-T-man5A5′), transformed into JM109, and sent to Shanghai Sangong for sequencing after enzyme digestion and identification. The 5' end mRNA sequence of Aus man5A was obtained.
Embodiment 3
[0042] Cloning of the 5' end regulatory sequence of embodiment 3β-mannanase gene
[0043] Using the processed A.usamii E001 genomic DNA as a template, the first round of PCR used T-PrimerF and Man5A-5R1 as primers, and the reaction conditions were: 94°C for 4min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C 60s), 72°C for 10min; the second round of PCR uses T-PrimerF and Man5A-5R2 as primers, and the reaction conditions are: 94°C, 4min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 60s), 72°C 10min. The two rounds of PCR products were analyzed by 1% agarose gel electrophoresis, the target band was recovered by tapping the gel and ligated with pUCm-T (pUCm-T-man5AP), transformed into JM109, and sent to Shanghai Sangong for sequencing after enzyme digestion and identification. Aus man5A 5' regulatory sequence.
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