DNA的稳定同位素探测,识别和特性的微生物能够利用特定的基板活跃的社区是一个独立的种植方法。基板同化富集重同位素的信息纳入到微生物生物量的标记原子。密度梯度超速离心检索下游分子生物学分析的DNA标记。
DNA稳定同位素探测(脱氧核糖核酸SIP的)是一个强大的技术,确定积极融入细胞生物量的微生物特别是碳基体和养分。因此,培养独立的技术已分配的代谢功能多样的社区居住的陆地和水生环境的广泛的一个重要方法。与稳定同位素标记化合物的环境样品的潜伏期后,提取核酸是密度梯度离心和随后的梯度分馏分离不同密度的核酸。氯化铯的DNA纯化检索随后的分子特性(如指纹识别,基因芯片,克隆库,宏基因组学)标签和不知名的DNA。这朱庇特的视频协议提供视觉密度梯度超速离心,梯度分馏和回收标记的DNA协议一步一步的解释。该协议还包括样品的SIP数据,并强调重要的提示和警告说,必须考虑,以确保成功的DNA – SIP的分析。
正确的设计是稳定同位素探测实验,获得上述背景不知名社区标记的DNA至关重要。样品孵育时间,底物浓度,培养条件(如养分,土壤水分含量),跨期和复制有关的注意事项进行了讨论别处 10,18,我们建议读者查阅这些出版物时,设计一个SIP孵化。目前的协议,这是值得评论从SIP梯度数据的解释有关的额外考虑。由于离心过程的性质,它是另外重要的,包括纯文化和母语的底物孵育样?…
The authors have nothing to disclose.
这项工作是支持的战略项目和发现资助自然科学和加拿大工程研究理事会(NSERC)JDN。
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Bromophenol Blue | Reagent | Fisher Scientific | BP115-25 | |
Cesium chloride | Reagent | Fisher Scientific | BP210-500 | |
Ethanol, reagent grade | Reagent | Sigma-Aldrich | 652261 | |
Ethidium bromide | Reagent | Sigma-Aldrich | E1510 | |
Hydrochloric acid | Reagent | Fisher Scientific | 351285212 | |
Linear polyacrylamide | Reagent | Applichem | A6587 | |
Polyethylene Glycol 6000 | Reagent | VWR | CAPX1286L-4 | |
Potassium Chloride | Reagent | Fisher Scientific | AC42409-0010 | |
Sodium Chloride | Reagent | Fisher Scientific | S2711 | |
Sodium Hydroxide pellets | Reagent | Fisher Scientific | S3181 | |
Tris base | Reagent | Fisher Scientific | BP1521 | |
Dark Reader | Equipment | Clare Chemical | DR46B | |
Microcentrifuge | Equipment | Eppendorf | 5424 000.410 | |
Nanodrop 2000 | Equipment | Fisher Scientific | 361013650 | |
Infusion pump | Equipment | Braintree Scientific | N/A | Model Number: BSP See www.braintreesci.com for ordering details. |
Tube sealer | Equipment | Beckman-Coulter | 358312 | |
Ultracentrifuge | Equipment | Beckman-Coulter | ||
Ultracentrifuge rotor | Equipment | Beckman-Coulter | 362754 | |
Ultraviolet light source | Equipment | UVP Inc. | 95-0017-09 | Any UV source will suffice |
Ultraviolet light face shield | Equipment | Fisher Scientific | 114051C | |
Butyl rubber stoppers, gray | Material | Sigma-Aldrich | 27232 | |
Centrifuge tubes | Material | Beckman-Coulter | 342412 | |
Hypodermic needle, 23 gauge, 2” length | Material | BD | 305145 | |
Microfuge tubes, 1.5 mL | Material | DiaMed | AD151-N500 | |
Open center seals, 20 mm diameter | Material | Sigma-Aldrich | 27230-U | |
Pasteur pipettes, glass | Material | Fisher Scientific | 13-678-6C | |
Pipet tips | Material | DiaMed | BPS340-1000 | Catalogue number is for 200 μl tips. 10 or 20 μl tips may be purchased from the same source |
Pump tubing 1.5 mm bore x 1.5 mm wall | Material | Appleton Woods | ||
Screw-cap tubes, 15 mL | Material | DiaMed | AD15MLP-S | |
Serum vials, 125 mL volume | Material | Sigma-Aldrich | Z114014 | |
Syringe, 60 mL | Material | BD | 309653 |