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Compared to several expensive RNA extraction kits, the following protocol provides an economic and simple method for researchers to extract yeast RNA. This method can achieve RNA quality that is sufficient for most northern blot studies in yeast.

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A Simple Preparation of RNA from Yeast by Hot Phenol for Northern Blot
用于Northern印记杂交实验的酵母RNA的提取-简易热苯酚法

分子生物学 > RNA > RNA 提取
作者: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
Vol 2, Iss 12, 6/20/2012, 6572 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.209

[Abstract] Compared to several expensive RNA extraction kits, the following protocol provides an economic and simple method for researchers to extract yeast RNA. This method can achieve RNA quality that is sufficient for most northern blot studies in yeast.

[Abstract] 与昂贵的RNA 提取试剂盒相比, 以下规程为研究者提供了一个经济简便的提取酵母RNA的方法。 这种方法所达到的RNA质量足以进行酵母northern blot研究。

Materials and Reagents

  1. W303a cell line
  2. Sodium acetate trihydrate (CH3CO2Na•3H2O) (Sigma-Aldrich, catalog number: 236500 )
  3. Phenol (C6H5OH) (Sigma-Aldrich, catalog number: P1037 )
  4. EDTA (Na2EDTA•2H2O) (Sigma-Aldrich, catalog number: ED2SS )
  5. Acetic acid (CH3COOH) (Sigma-Aldrich, catalog number: 320099 )
  6. Chloroform (CHCl3) (Sigma-Aldrich, catalog number: 472476 )
  7. 8-hydroxyquinoline (C9H7NO) (Sigma-Aldrich, catalog number: 252565 )
  8. NaCl (Thermo Fisher Scientific, catalog number: S641-500 )
  9. Synthetic complete (SC) medium
  10. SDS (Sigma-Aldrich, catalog number: L3771 )
  11. Isoamyl alcohol (Sigma-Aldrich, catalog number: W205710 )
  12. Ethanol (Thermo Fisher Scientific, catalog number: 64-17-5 )
  13. DEPC water
  14. Phenol-AE buffer (see Recipes)
  15. Phenol: CHCl3 /AE-Na (see Recipes)
  16. CHCl3: Isoamyl alcohol (24:1) (see Recipes)

Equipment

  1. Standard bench-top centrifuge
  2. Microfuge
  3. Shaker
  4. 1.5 eppendorf tube
  5. Liquid nitrogen

Procedure

  1. Prepare reagents according to recipes. Note that everything is in DEPC water.
  2. Inoculate W303a cells expressing different TOR1-RR variants in 2 ml SC medium overnight.
  3. Subculture the cells starting from OD600=0.1 in 10 ml SC media, shake vigorously at 30 °C, 300 rpm for around 4-6 h until OD600=0.4-0.5.
  4. Collect the cells by spinning down without freezing on ice. Discard supernatant.
  5. Re-suspend cells with 1 ml water and transfer to a 1.5 eppendorf tube, quickly spin down at 3,000 x g for 15 sec.
  6. Re-suspend cell pellet in 400 µl of AE buffer at room temperature.
  7. Add 40 µL 10% SDS (final around 1%) and vortex briefly at room temperature (RT).
  8. Immediately add 500 µl hot phenol/AE (put in 65 °C for 10 min before use), vortex vigorously for 1 min.
  9. Incubate at 65 °C for 5 min. Briefly vortex every 30 sec.
  10. Immediately freeze by dumping into liquid nitrogen (labels won’t get off).
  11. Wait to thaw at RT (put in 30 °C to thaw may crack the tube).
  12. Centrifuge for 10 min on a standard laboratory microfuge at 20,000 x g at RT.
  13. Transfer around 400 μl supernatant to a new eppendorf tube. Recycle the lower phenol fraction carefully following the chemical safety protocol in your laboratory.
  14. Add equal volume (400 μl) phenol: CHCl3/AE-Na. Vortex vigorously for 1 min at RT.
  15. Spin down at 20,000 x g for 5 min in a standard laboratory microfuge.
  16. Transfer supernatant (around 350 μl) to a fresh 1.5 ml eppendorf tube.
  17. Add CHCl3: isoamyl alcohol (24:1). Vortex vigorously for 1 min at RT.
  18. Transfer aqueous supernatant to fresh 1.5 ml microfuge tube. If white cloudy precipitate is observed between aqueous phase and organic phase, repeat steps 17-18.
  19. Add 1/10 volume of 3 M NaOAc (pH 5) and vortex vigorously. Add 2.5 volumes of ethanol. Vortex again.
  20. Place at -20 °C for at least 30 min.
  21. Spin down in the microfuge at 20,000 x g, 15 min at 4 °C. RNA pellet is usually visible.
  22. Add ice-cold 75% EtOH, place at 4 °C for around 10 min. Vortex and spin down on microfuge 20,000 x g, 15 min at 4 °C.
  23. Discard supernatant. Suck out the liquid droplets in the tube.
  24.  The white RNA pellet will turns clear when it dries out. Add 30-50 µl ddH2O (DEPC) immediately after it becomes clear.
  25. Do not let the RNA over-dry, which will make it difficult to dissolve. If RNA pellet is over-dry, dissolve RNA at 37 °C for 30 min.
  26. Store RNAs at -80 °C for more than 2 months.

Recipes

  1. Phenol-AE buffer
    Make Acetate-EDTA(AE) buffer
    50 mM NaOAc
    10 mM EDTA (pH 5.0)
    Liquefied phenol equilibrated with equal volume of AE buffer. Store at 4 °C.
  2. Phenol: CHCl3 /AE-Na
    Make AE-Na buffer
    10 mM NaOAc
    2 mM EDTA
    100 mM NaCl (pH 6.0)
    50% phenol/AE
    50% CHCl3
    0.25% 8-hydroxyquinoline
    Store at 4 °C.
  3. CHCl3: Isoamyl alcohol (24:1)

Acknowledgments

This protocol was adapted from and used in Wei and Zheng (2009) and Wei et al. (2009).

References

  1. Wei, Y. and Zheng, X. F. (2009). Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell Cycle 8(24): 4085-4090.
  2. Wei, Y., Tsang, C. K. and Zheng, X. F. (2009). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J 28(15): 2220-2230.

材料与试剂

 

1.         三水醋酸钠 (CH3CO2Na·3H2O, Sigma 公司,货号# 236500)

2.         苯酚 (C6H5OH, Sigma公司,货号# P1037)

3.         EDTA (Na2EDTA·2H2O, Sigma公司,货号# ED2SS)

4.         乙酸 (CH3COOH, Sigma 公司,货号# 320099)

5.         三氯甲烷 (CHCl3, Sigma 公司,货号# 472476)

6.         8-羟喹啉 (C9H7NO Sigma公司,货号# 252565)

7.         氯化钠 (Fisher 科学,货号# S641-500)

8.         合成完全 (SC) 培养基

9.         SDS (Sigma公司,货号#  L3771)

10.     异戊醇(Sigma 公司,货号#  W205710)

11.     乙醇 (Fisher 科学,货号# 64-17-5)

 

设备

 

1.         离心机

2.         系列台式微量离心机

3.         混合器

4.         1.5 ml eppendorf

5.         液氮

 

程序

 

1.         依据配方制备试剂,注意一切试剂配制均使用DEPC水。

2.         接种表达不同TOR1-RR 变体的W303a cell 2 mL 合成完全培养基,过夜培养。

3.         10 mL 完全合成培养基中次培养初始OD6000.1的细胞, 30 °C, 300 rpm 摇晃培养 4-6 小时, OD600达到0.4~0.5.

4.         缓慢旋转收集细胞 ,不需置于冰上, 弃上清。

5.         1mL 水重悬细胞并转移到1.5ml eppendorf, 快速3000g 离心15秒。

6.         室温下重悬细胞沉淀于400 μL AE 缓冲液中。

7.         加入 40 μL 10% SDS (至终浓度大约1%) 并且室温下短暂漩涡。

8.         立即加入 500 μL 热苯酚/AE (使用前置65°C10分钟), 剧烈震荡1分钟。

9.         65°C 孵育 5 分钟,  30 秒短暂漩涡。

10.     置于液氮中立即冷冻. (标签不能脱落)

11.     室温下等待融解 (置于30 °C 融解可能导致管破裂)

12.     室温下置于系列台式微量离心机中20000g转速离心10 分钟。

13.     转移大约 400 μl 上清于新的eppendorf管中。依据化学安全规程回收底部残留苯酚。

14.     加入等体积 (400 μl) 苯酚: 三氯甲烷/AE-Na.,室温下剧烈漩涡震荡1分钟。

15.      系列台式微量离心机中20000g缓慢旋转5分钟。

16.     转移上清 (大约350 μl)到一个新的 1.5 mL eppendorf 管中。

17.      往管中加入三氯甲烷: 异戊醇 (24:1),室温下剧烈漩涡震荡1分钟。

18.     转移水相上清到新鲜的1.5 ml 离心管。如果在水相和有机相中观察到白色雾状沉淀,重复 (17)-(18) 步骤。

19.     往管加入 1/10 体积3 M NaOAc, pH 5, 剧烈漩涡震荡。 加入2.5 体积的乙醇,再次漩涡。.

20.     将管置于 -20°C 至少 30 minutes.

21.      4°C下在系列台式微量离心机中20000g,减慢旋转15 分钟,通常RNA 沉淀是可见的。

22.     加入冷冻的 75% 乙醇, 置于 4°C 大约 10 分钟,漩涡并且在4°C于系列台式微量离心机20000g减慢旋转15分钟。

23.     弃上清, 吸出管中液滴。

24.     当干燥后RNA沉淀变得清晰。在RNA沉淀变得清晰之后立即加入30-50 μL ddH2O (DEPC)

25.     不要让RNA 过于干燥,这样会使RNA难于溶解。如果RNA 沉淀过于干燥,37°C溶解 RNA30分钟。

26.     RNA-80°C 可保存2个月以上。

 

配制溶液

 

1.         苯酚-AE 缓冲液:

配制醋酸盐-EDTA(AE)缓冲液: 50 mM NaOAc, 10mM EDTA pH5.0, 用等体积AE缓冲液去平衡液相苯酚,于4°C储存。

2.         苯酚: 三氯甲烷 /AE-Na:

配制AE-Na 缓冲液: 10 mM NaOAc, 2 mM EDTA, 100 mM NaCl pH6.0.  50% 苯酚/AE, 50% 三氯甲烷 + 0.25% 8-羟喹啉,并于 4°C保存.

3.         三氯甲烷: 异戊醇(24:1)

 

参考文献

 

1.         Wei Y., Zheng X.F. (2009). Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell Cycle 8(24): 4085-90. 

2.         Wei Y., Tsang C.K., Zheng X.F. (2009). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO Journal 28(15): 2220-30. 

 

 

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How to cite this protocol: Wei, Y. (2012). A Simple Preparation of RNA from Yeast by Hot Phenol for Northern Blot. Bio-protocol 2(12): e209. DOI: 10.21769/BioProtoc.209; Full Text



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