Pseudo monads pseudoalcaligenes gene promoter
A Pseudomonas, promoter technology, applied in genetic engineering, plant genetic improvement, biochemical equipment and methods, etc., can solve problems such as low transcription efficiency
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Embodiment 1
[0047] Pseudomonas pseudoalcaligenes total DNA extraction
[0048] Pseudomonas pseudoalcaligenes was activated overnight in 2ml LB, inoculated in 50ml broth medium at a ratio of 1:100, and cultured for 12hr. Put the bacteria into a 50ml centrifuge tube, centrifuge at 3500rmp for 10min, remove the supernatant, turn the centrifuge tube upside down on a paper towel and let the supernatant flow out at night; add a small amount of lysate to suspend the bacteria, and then add lysate to a total volume of 4ml , add 1ml SDS, mix well, 60°C for 10min; add equal volumes of phenol, phenol / chloroform extract, and chloroform to extract once; take the supernatant, add 1 / 10 volume of 3M potassium acetate (pH4.8), mix Mix well, then add 2 times the volume of 100% ice-cold ethanol along the tube wall, shake gently until a filamentous precipitate appears; centrifuge at 2000rpm for 5 minutes, remove the supernatant; add 1ml of 70% ethanol, wash the precipitate once, and let it dry naturally at ro...
Embodiment 2
[0050] produce DNA fragments
[0051] Use Pseudomonas pseudoalcaligenes total DNA extract (1 μ g) to pass through in (100 μ l volume) 100 mM NaCl, 50 mM Tris-HCl (pH7.5), 10 mM MgCl 2 , Dithiothreitol in 1mM was digested with Sau3A I [Bao Biological Engineering (Dalian) Co., Ltd. (TAKARA)] to generate a DNA fragment of Pseudomonas-like alcaligenes. The mixture was incubated at 37°C for 120 minutes and quenched with 10 mM EDTA. The Sau3A1 fragment was precipitated from the digestion mixture with ethanol and washed with ethanol.
Embodiment 3
[0053] Transformation of E. coli and selection of transformants
[0054] The Sau3AI viral fragment produced as described in Example 1 was cloned into plasmid pSUPV4 by the shotgun cloning method described in Sambrook et al., 1989, in Cloning. Plasmid promoter probe vector pSUPV4 (AP r ) was constructed by the Laboratory of Molecular Biology, School of Life, Sichuan University (Zhang Yizheng et al. Journal of Sichuan University (Natural Science Edition), 1998, 35(2): 263-267.). The map of pSUPV4 is shown in Figure 1 . DNA preparation, fill-in reactions and ligation were performed as described by Sambrook et al. (supra) or according to the manufacturer's instructions.
[0055] The DNA fragment of Pseudomonas pseudoalcaligenes was ligated with 1 μg of pSUPV4 pre-treated with BamHI and calf intestinal alkaline phosphatase (CIP) to generate recombinant plasmids PA1; PA2; PA3; PA4: PA5; PA6; PA7; PA8; PA9 . These 9 are related to the heterologous kanamycin resistance marker gene...
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