Summary

DNA的稳定同位素探测(DNA的SIP)

Published: August 02, 2010
doi:

Summary

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。

Abstract

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。

Protocol

1。试剂的制备 DNA – SIP的要求,应在实际过程提前准备试剂的使用。本节列出每种试剂的准备方向,并从以前的SIP协议进行修改。 氯化铯(CSCL)的解决方案,准备SIP的梯度逐渐溶解603.0克,中海集运(DDH 2 O)的蒸馏水和去离子水到终体积为500 mL -准备一个7.163中号铯的解决方案。小心不超过500毫升!略有边搅拌变暖的解决方案将有助于溶解所有的CSCL。分装在?…

Discussion

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?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

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。

Materials

Material Name Tipo 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  

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Citazione di questo articolo
Dunford, E. A., Neufeld, J. D. DNA Stable-Isotope Probing (DNA-SIP). J. Vis. Exp. (42), e2027, doi:10.3791/2027 (2010).

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