RNA Isolation from Synechocystis
集胞藻细胞RNA的提取
发布: 2015年03月20日第5卷第6期 DOI: 10.21769/BioProtoc.1428 浏览次数: 23422
评审: Maria SinetovaClaudia CatalanottiAksiniya Asenova
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参见作者原研究论文
The authors used this protocol in:
Aug 2014
Abstract
The protocol describes the procedure of total RNA isolation from cells of the cyanobacterium Synechocystis sp. PCC 6803. This protocol is also applicable to Synechococcus elongatus PCC 7942 and PCC 6301, Thermosynechococcus vulcanus, and other unicellular and filamentous species of cyanobacteria that do not have thick polysaccharide-containing outer layers. For the latter, Trizol-containing protocols should be adapted. The yield of RNA depends on optical density of cyanobacterial culture and may reach up to 10-20 µg of total RNA per 1 ml of cell culture. RNA isolated by this method can be used for Northern blot hybridization, RT-qPCR, microarrays and Next Generation Sequencing.
Keywords: Synechocystis (集胞藻)
Cyanobacteria (蓝藻)
RNA isolation (RNA的分离)
RNA quality (RNA的质量)
Hot phenol (热酚)
Materials and Reagents
- Cyanobacterial culture (50 ml) (OD750 ~ 2) grown in BG11 medium (Rippka, 1988)
- 95% ethanol for molecular biology (keep at -20 °C)
- 70% ethanol for molecular biology (30 ml of RNAse free water and 70 ml 95% ethanol, keep at -20 °C)
- 70% ethanol for sterilization
- Phenol BioUltra (for molecular biology, TE-saturated, ~73%) (Sigma-Aldrich, catalog number: 77607 ) (keep at 4 °C)
- 1 M Tris-HCl (Life Technologies, catalog number: 15568-025 )
- 0.5 M EDTA-Na2 (pH 8.0) (Life Technologies, catalog number: AM9260G )
- RNase-free autoclaved water
- Chloroform (Sigma-Aldrich, catalog number: C2432 ) (keep at 4 °C)
- 10 M lithium chloride solution (Fluka, catalog number: 83268 ) (chill on ice before use)
- RNAse-free DNAse
- Agarose for molecular biology (Sigma-Aldrich, catalog number: A9539 )
- TAE electrophoresis buffer (Sigma-Aldrich, catalog number: T9650 )
- RNA ladder (Life Technologies, InvitrogenTM, catalog number 15620-016 )
- TE buffer (see Recipes)
- 50/100 TE buffer (see Recipes)
- 10 M LiCl solution in water (see Recipes)
- Cell fix solution (see Recipes)
Equipment
- Saran wrap (any type)
- Autoclave bag (Sigma-Aldrich, catalog number: Z692212 )
- Safe-Lock tubes (2.0 ml) (Eppendorf, catalog number: 0030 120.094 )
- 50 ml plastic conical polypropylene tubes (sterile, nuclease-free, nonpyrogenic, autoclavable) (Life Technologies, catalog number: AM12501 or Corning, Falcon )
- Autoclave
- Refrigerated centrifuge with bucket rotor that fits 50 ml conical tubes (Eppendorf, model: 5804 R and Swing-bucket rotor A-4-44 with 50 ml Falcon adapters)
- Refrigerated microcentrifuge preset to 4 °C (Eppendorf, model: 5415 R )
- Water bath preset to 65 °C
- Subzero refrigerators: -20 °C and -70 °C
- Fume hood
- Ice bath
- Multi tube automated vortex (for example, Micro tube mixer MT-400, Tomy Digital Biology)
- UV-spectrophotometer (preferably, Nanodrop)
- Agarose gel electrophoresis equipment (VWR Mini Gel II Complete Horizontal Electrophoresis System, catalog number: 95043-650)
Note: Workplace, pipettes, gloves etc. should be cleaned with 70% ethanol before RNA isolation procedures. Conical tubes, Eppendorf tubes, pipette tips should be sterilized by autoclaving in autoclave bags before use.
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Mironov, K. S. and Los, D. A. (2015). RNA Isolation from Synechocystis. Bio-protocol 5(6): e1428. DOI: 10.21769/BioProtoc.1428.
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分类
微生物学 > 微生物遗传学 > RNA
分子生物学 > RNA > RNA 提取
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