Not all cyanobacterial strains produce toxins. However, the toxin-producing strains cannot be distinguished from the nontoxin-producing strains by traditional light microscopy, commonlyused to monitor water bodies. An alternative for the differentiation of potentially toxic strains from nontoxic strains is to use molecular methods to detect the presence of toxin biosynthetic genes. Such methods are already available and could be used for the detection and identification of potential microcystin and nodularin producers present in environmental samples (Attogene catalog number NA2024).
Screening for the toxin itself, can be very costly. In turn, real time PCR for the detection of a gene region responsible for assembling in cyanobacterial strains and environmental samples can be a key indicator for the prescense of cyanobacteria capable of expressing the aetokthonotoxin toxin. Attogen has thus, designed primer pairs and probes targeting a the conserved gene region in order to enable the amplification and detection of several producer genera using real time PCR. Screening for the toxin genes can save significant costs and act as a triage for samples needing to be analyzed for the toxin itself.
Cyanobacterial neurotoxin aetokthonotoxin (AETX), a peculiar pentabrominated biindole alkaloid implicated in fatal Vacuolar Myelinopathy. This neurodegenerative disease was first recorded in 1994 during an outbreak of bald-eagle poisonings at De Gray Lake in Arkansas, USA. AETX was experimentally confirmed to be produced by the true branching heterocytous cyanobacterium Aetokthonos hydrillicola. The production of AETX is dependent on bromide (Br−) availability, and likely linked to its hyper-accumulation by the host plan. Thus regular monitoring of A. hydrillicola (accompanied by assessment of Br− and AETX levels) is highly advisable to predict the possible threat of further VM outbreaks.
The cyanobacterial AetA gene which encodes the unique FAD-dependent halogenase involved in the pathway for AETX synthesis has been adapted to develop a -aetokthonotoxin specific quantitative PCR (qPCR) assay.
Real time qPCR kit for AetA gene
For screening aetokthonotoxin gene cluster
Use in combination with Attogene Algae DNA isolation kit
K-PullG6
SKU: 700004329
100/200 assays per kit
| Content: | 100 / 200 assays per kit |
| Shipping Temperature: | Ambient |
| Storage Temperature: | Short term stability: 2-8oC, Long term stability: See individual component labels |
| Stability: | > 2 years under recommended storage conditions |
| Analyte: | Pullulanase/Limit-Dextrinase |
| Assay Format: | Spectrophotometer |
| Detection Method: | Absorbance |
| Wavelength (nm): | 400 |
| Signal Response: | Increase |
| Limit of Detection: | 0.18 U/mL for pullulanase preparations (50-fold dilution) 0.01 U/g for limit dextrinase in milled malt |
| Reproducibility (%): | ~ 3% |
| Total Assay Time: | ~ 10 min (Pullanase), ~ 30 min (Limit-Dextrinase) |
| Application examples: | Assay of microbial pullulanase preparations. Measurement of limit-dextrinase in malt extracts. |
| Method recognition: | Novel method |
PullG6 assay for the measurement of pullulanase employs a water soluble defined substrate, namely 4,6-O-benzylidene-4-nitrophenyl-63-α-D-maltotriosyl-maltotriose (BPNPG3G3), coupled with the ancillary enzymes α-glucosidase and β-glucosidase. Upon hydrolysis of the substrate at the 1,6-α-linkage by pullulanase or limit-dextrinase, the released 4-nitrophenyl-β-maltotrioside is immediately hydrolysed to glucose and 4-nitrophenol by the concerted action of the α-glucosidase and β-glucosidase enzymes in the reagent mixture. The reaction is terminated and phenolate ions are developed by addition of dilute alkali. The absorbance is read at 400 nm and the value obtained correlates directly with pullulanase activity.
Explore more of our assay kit products for enzyme activity measurement.
Advantages
The HiPure Midi system provides a fast, simple, and cost-effective plasmid DNA midiprep method for routine molecular biology laboratory applications. HiPure Midiprep Kits use silica membrane technology to eliminate the cumbersome steps associated with loose resins or slurries. Plasmid DNA purified with Mini Kits is immediately ready for use. Phenol extraction and ethanol precipitation are not required, and high quality plasmid DNA is eluted in a small volume of Tris buffer or water.
Specifications
| Features | Specifications |
| Main Functions | Isolation up to 500µg endotoxin-free plasmid DNA from 25-50ml bacterial culture. Recommend for High copy vector, Low elution volume, High concentration |
| Applications | Cell transfection, animal injection, etc. |
| Purification method | Midi spin column |
| Purification technology | Silica technology |
| Process method | Manual (centrifugation or vacuum) |
| Sample type | High copy plasmid vector |
| Sample amount | 25-50ml LB |
| Yield | 10-500μg |
| Elution volume | ≥0.1ml |
| Time per run | ≤60 minutes |
| Liquid carrying volume per column | 4ml |
| Binding yield of column | 250μg |
The HiPure Plasmid procedure is based on alkaline lysis of bacterial cells followed by adsorption of DNA onto silica in the presence of high salt. The unique silica membrane used in the kit completely replaces glass or silica slurries for plasmid DNA minipreps. The procedure consists of 3 basic steps: Preparation and clearing of a bacterial lysate by alkaline method，then transfer the supernatant to column to bind DNA. After washing proteins and other impurities, nucleic acid was finally eluted with low-salt buffer (10mm Tris, pH9.0, 0.5mm EDTA).
Advantages
Kit Contents
| Contents | P123102 | P123102B |
| Purification Times | 50 Preps | 50 Preps |
| RNase A | 30 mg | 30 mg |
| Buffer E1 | 150 ml | 150 ml |
| Buffer E2 | 150 ml | 150 ml |
| Buffer E3 | 150 ml | 150 ml |
| Buffer E4 | 150 ml | 150 ml |
| Buffer E5 | 150 ml | 150 ml |
| Buffer PW2* | 50 ml | 50 ml |
| Elution Buffer | 30 ml | 30 ml |
| MaxPure EF Mini Column | 50 | 50 |
| 2 ml Collection Tubes | 50 | 50 |
| Lysate Clear Midi Syringe | 50 | 50 |
| Extend Tubes | 50 | 50 |
| 50ml Centrifuge Tubes | 50 | |
| Support Tubes | 50 |
Storage and Stability
The kit components can be stored dry at room temperature (15-25°C) and are stable for at least 18 months under these conditions. If any precipitates form in the buffers, warm at 37℃ to dissolve. After addition of RNase A and optional LyseBlue reagent, Buffer P1 is stable for 6 months when stored at 2–8°C.
For any technical problems or customized products, please contact us.
F&Q about Endotoxin-free Plasmid Extraction Kit — P1156 ←click here
The HiPure Midi system provides a fast, simple, and cost-effective plasmid DNA midiprep method for routine molecular biology laboratory applications. HiPure Midiprep Kits use silica membrane technology to eliminate the cumbersome steps associated with loose resins or slurries. Plasmid DNA purified with Mini Kits is immediately ready for use. Phenol extraction and ethanol precipitation are not required, and high quality plasmid DNA is eluted in a small volume of Tris buffer or water.