Nostoc high-efficiency cracking phycophage YongM and application thereof
A technology of cyanophage and Nostoc, which is applied in the field of high-efficiency and potent cyanophage of Nostoc, can solve the problems of changing the photosynthesis center of the host, reducing the incidence of photosynthesis, and affecting the primary productivity of water bodies, achieving good development prospects and high replication rate, the effect of high infection rate
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Embodiment 1
[0033] Preparation of cyanophage YongM
[0034] (1) Cultivation of Nostoc sp.FACHB-596
[0035] Take 5mL of Nostoc sp.FACHB-596 algae liquid, dilute it into a conical flask filled with 500mL of BG11 liquid medium at a volume ratio of 1:100, and place it at a temperature of 25°C, with a light intensity of 2000lx and a light-dark cycle of In a 12h:12h light incubator, the logarithmic phase FACHB-596 algae liquid can be obtained in about 10 days.
[0036] (2) Enrichment and screening of cyanophages
[0037] Water sample treatment: Collect surface water samples from various places in Dianchi Lake, Yunnan, and bring them back to the laboratory for processing as soon as possible. Water samples were centrifuged at 12,000 g for 20 minutes at 4°C to remove precipitates. Take the supernatant for the experiment.
[0038]Take a number of sterile Erlenmeyer flasks corresponding to the number of samples, and add to each Erlenmeyer flask: 80mL of the above-treated water sample supernatan...
Embodiment 2
[0055] Morphological observation of cyanophage YongM
[0056] Take the cyanophage-algal culture lysate separated and purified in Example 1 and centrifuge at low speed (4°C, 5000g, 5min) to discard the precipitate, distribute the supernatant evenly to two tubes, and inject slowly and gently from the bottom with a syringe 5-10 mL of 20% (w / v) sucrose solution was centrifuged at 35,000 g at 4°C for 1 h, and the supernatant was discarded. Gently add 0.01M PBS, discard PBS, add 0.01M PBS again and place at 4°C overnight to suspend the precipitate. Use a pipette gun to take a drop of cyanophage suspension onto the copper grid, let it stand for 10 minutes, and then use neutral filter paper to absorb excess water from the side. Drop a drop of 3% uranyl acetate on the copper grid, and after staining for 20 seconds, use neutral filter paper to absorb the staining agent from the side. After standing for 10 minutes to dry, the morphology of cyanophages was observed with a transmission e...
Embodiment 3
[0059] Sequence Alignment of DNA Polymerase Genes of Phage YongM
[0060] Genome extraction: Add DNase I and RNase A to the cyanophage YongM suspension to a final concentration of 1 μg / mL, digest overnight at 37°C, and inactivate at 80°C for 15 minutes. Lysis solution (0.5% SDS, 50 μg / mL proteinase K, 20 nM EDTA, both final concentrations) was added to the system, and incubated at 56° C. for 1 h. Add an equal volume of equilibrated phenol, and after gentle shaking, centrifuge at 10000g for 5min at 4°C. The upper layer was collected, and an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) was added, and after gentle shaking, it was centrifuged at 10,000 g for 5 min at 4°C. Collect the upper layer liquid, add an equal volume of chloroform, mix well, centrifuge at 10000g for 5min, collect the upper layer liquid, and repeat 2 times. Add an equal volume of isopropanol, place at -20°C for at least 30min, centrifuge at 10,000g for 20min at 4°C, and wash the precipitate t...
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