A dna sequence and its application
A DNA sequence and nucleotide sequence technology, applied in the field of genetic engineering, can solve problems such as tediousness, increased production costs, and cell death
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Embodiment 1
[0042] Embodiment 1: Construction of BZIP transcription factor overexpression vector
[0043]BZIP transcription factor of the present invention contains 856 amino acids, uses SMART website and NCBI website to analyze the protein sequence of putative BZIP transcription factor, finds that its BZIP transcription factor contains 2 structural domains: FBPcase structural domain (12-399), BZIP Domain (499-543) ( figure 1 A).
[0044] Genomic DNA of Nannochloropsis was extracted (Meiji DNA Extraction Kit). Primers (Shanghai Sangon Bioengineering Co., Ltd.) were purified by PAGE and diluted to 20 μM when used. The primer pair Na07F, Na08R (Table 1) was used to amplify the sequence of the BZIP transcription factor with the genome of Nannochloropsis as a template, the full length of which is 4057bp.
[0045] Table 1. Related primers
[0046]
[0047] Table 2. PCR reaction system (20μl)
[0048]
[0049] The reaction conditions are: pre-denaturation: 94°C, 2min.
[0050]
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Embodiment 2
[0054] Embodiment 2: Transformation of Nannochloropsis and screening of transformed algae
[0055] In the first step, the recombinant overexpression vector (pna03-[BZIP]TF) was linearized using ScaI restriction endonuclease (TAKARA), incubated for 8h, and then purified using a DNA fragment purification kit (takara); the second step , Transform the linearized recombinant overexpression vector by electric shock, the method is as follows: 4400rpm, 10min centrifugation to collect algae cells, use 375mmol sterile sorbitol to wash 3 times, remove the seawater inside as much as possible, finally, use 150μl sterile sorbitol The solution was used to resuspend the algal cells, add 30ug of pre-cooled silicon essence DNA (heated at 95 degrees for 1min), and 3ug of linearized recombinant overexpression vector, let it stand on ice for 10min, and use an electric shock device for electric shock, the conditions are as follows: 2.2 kv, the capacitance is 50Uf, and the capacitance is 600ohms. I...
Embodiment 3
[0056] Example 3: Verification of Transformed Algae
[0057] The first step is to verify at the molecular level, centrifuge 100ml of transformed algae (BZIP-TF1 and BZIP-TF2) and 100ml of wild-type Nannochloropsis at 4400rpm for 10min, collect the microalgae, and extract genomic DNA (Meiji DNA Extraction Kit ), utilize primer Na01 and Na02 to carry out PCR, PCR condition, system refer to embodiment 1, the electrophoresis band of 4200bp has occurred in transformed algae (such as figure 2 shown), but not in the control algae; in the second step, the electrophoretic bands were recovered by cutting the gel, sequenced with primer Na01, and blasted on the NCBI network. image 3 The results showed that the 4057bp electrophoresis band was indeed the gene sequence of BZIP transcription factor. It shows that the recombinant overexpression vector (pna03-[BZIP]TF) is successfully integrated into the genome of microalgae.
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