欢迎您, 登录 | 注册

首页 | English

X
加载中

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

[Bio101] RNA Extraction from Yeast
[Bio101] 酵母RNA的提取

分子生物学 > RNA > RNA 提取
作者: Wei Zheng
Wei ZhengAffiliation: Keck Biotech Services, Yale University, New Haven, USA
For correspondence: wei.zheng.madison@gmail.com
Bio-protocol author page: a10
10/5/2011, 4932 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.138

[Abstract]

[Abstract]

Materials and Reagents

  1. DEPC treated water
  2. Phenol (TE)/Chloroform (1:1)
  3. 3 M NaAc (pH 5.2)
  4. NaOAc
  5. 10 mM EDTA
  6. 10% SDS
  7. EtOH
  8. Hydroxyquinoline
  9. Complete buffer A (see Recipes)
  10. Buffer A phenol or RNA phenol (see Recipes)
  11. TE phenol (see Recipes)

Equipment

  1. RNase free tube
  2. RNase-free plastic ware
  3. Water bath

Procedure

  1. Harvest Cells
    1. Grow cells to OD600 0.4-0.6 (mid-log phase).
    2. Spin down 10 ml cell cultures for each sample.
    3. Resuspend in 1 ml DEPC treated water, and transfer to RNase free tubes (Note: screw caps are preferred, since snap-caps turns to pop-open in hot bath). 
    4. Spin down cells, pour off supernatant, freeze at -80 °C or use right away.

  2. RNA extraction
    1. Prepare solutions:
      1. Make 0.5 ml buffer A per sample + 1. Aliquot needed amount into RNase-free plastic ware. Add 1% DEPC just before use. Keep at room temp (RT).
      2. Make 1.2 ml Phenol A per sample + 1. Aliqout phenol into RNase free plastic ware. Warm to 65 degrees.
      3. Make 0.5 ml Phenol (TE)/Chloroform (1:1). Aliquot amount needed into RNase free plastic ware. Store at RT.
      4. Prepare RNase free 3 M NaAc (pH 5.2), EtOH, 70% ETOH, and water.
    2. Remove cells from -80 °C and immediately add 0.5 ml buffer A.
    3. Add 600 μl of Phenol A (pre-warm and equilibrated to 65 °C). Add to all samples at once, putting tubes in 65 °C bath. Vortex two tubes at a time for 5 sec each. Keep rotating through tubes for 6 min. 
    4. Spin down tubes for 2 min. Place in water bath.
    5. Carefully remove the phenol layer using an RNase-free blue tip. You will be leaving the aqueous layer in the tube. Store in water bath while finishing extractions and add 600 μl of fresh phenol A.
    6. Vortex tubes for another 6 min. Spin down 2 min. While spinning, label new set of RNase-free Epi tubes (snap-cap). Add 200 μl water and 500 μl Phenol (TE)/Chloroform to each tube.
    7. Take off the aqueous layer and transfer to tubes containing water and phenol/Chloroform. Store in water bath.
    8. Vortex each tube for about 5 sec then spin down for 2 min. While spinning label new set of Epi tubes.
    9. Put tubes back into water bath. Take off top aqueous layer making sure not to get any of the bottom layer (Note: this is important to avoid contaminating DNA, you have to sacrifice ~ 20 μl liquid near the interface). Store in water bath until all are complete.
    10. Add 50 μl RNase-free 3 M NaAc and 1 ml EtOH. Mix. Store on ice 15 min.
    11. Spin down at 4 °C for 15 min.
    12. Wash with 1 ml 70% EtOH(RNase-free). Spin down for 10 min at 4 °C.
    13. Pour off as much EtOH as you can, then add 400 μl water. Let sit in water bath for about 5 min. Add 40 μl NaAc and 800 μl EtOH, mix, and store on ice for 15 min.
    14. Wash with 70% EtOH as before, let dry inverted on paper towel under heat lamp.
    15. Resuspend in 50 μl sterile water (25 μl if original culture volume was under 5 ml).
    16. Let sit in water bath for 15 min. Vortex, spin down.
    17. Dilute 5 μl of RNA into 495 μl water. Determine absorbance at A260 and A240.

Notes

  1. Always wear gloves.
  2. Always use RNase-free tubes, tips, plastic-ware, and solutions. 
  3. Always aliquot stock solutions from RNA shelf into an RNase-free container so you do not contaminate the stock.
  4. When handling RNA, either keep tubes in ice bucket or water bath that is over 50 °C.

Recipes

  1. Complete Buffer A
    a.  Buffer A stock
         50 mM NaOAc
         10 mM EDTA
         Add 16.7 ml 3 M NaOAc (pH 5.2) to 20 ml 0.5 M EDTA to 963.3 ml water.
         Add 0.1 % DEPC, stir O/N, autoclave.
    b.  Complete Buffer A (user solution)
         Add 100 ml 10% SDS to 900 ml of Buffer A stock.
         Add 1 % DEPC just before use.
  2. Buffer A phenol or RNA phenol
    Saturate phenol with Buffer A stock.
    Add 0.1% hydroxyquinoline.
  3. TE Phenol
    Chloroform: Saturate phenol with 10 mM Tris-HCI (pH 8.0) and 1 mM EDTA.
    Use 50% TE-Phenol and 50% chloroform.

试剂与配方

 

1.        完全缓冲液A

1)         缓冲液A 母液:50 mM 醋酸钠, 10 mM EDTA,加16.7 ml 3 M 醋酸钠,pH 5.220 ml 0.5 M EDTA 963.3 ml水中。加0.1% DEPC搅拌过夜。高压蒸汽灭菌。

2)         完全缓冲液A (使用液) 100 ml 10%SDS900 ml的缓冲液A母液中。使用前加1% DEPC

2.         缓冲液A RAN :带缓冲液A母液的饱和。加0.1%羟基喹啉。

3.        TE:氯仿:饱和10 mM Tris-HCl pH 8.01mM EDTA。用50% TE-50%氯仿。

 

步骤

 

1.        采集细胞

1)         生长细胞OD600 0.4 -0.6 (mid-log phase).

2)         每样品缓慢加入10 ml细胞液

3)         重新悬于1 ml DEPC 处理的水中,转移到不含RNase的样品管(注:带拧盖的管更好,因为扣盖在热水浴中容易磞开)

4)         沉淀细胞,倒掉上清,在 -80°C冷冻保存或立即使用。  

2.        提取

1)         准备溶液

a.         每个样品需要 0.5 ml 缓冲液A + 1份需要的数量放在不含RNase的塑料容器内。用时加1%DEPC,在室温下存放。

b.        每个样品需要 1.2 ml A + 1放在不含RNase的塑料容器内。加热至65

c.         需要 0.5 ml (TE)/氯仿(11) 1份所需的数量放在不含RNase的塑料容器内,室温下存放。

d.        需要准备不含RNase3 M 醋酸钠 (pH 5.2), 乙醇, 70%乙醇和水。

2)         -80°C取出细胞并立即加0.5 ml缓冲液A

3)         加入 600μlA (预热并平衡至65°C)。马上加到所有测试样品中,把管放入65°C水浴中。振荡5秒,一次两管同时振荡。共维持振6分钟。

4)         缓慢旋转达2分钟。放于水浴。

5)         用不含RNase的物体小心除去层。要把水层留在管内。存放于水浴,完成提取并加入600μl的新鲜A

6)         样品管再次振荡6分钟。离心2分钟。离心间隙,标记一套新的不含RNaseEP管。每管中加入200 μl水和500 μl苯酚/氯仿。

7)         取出水层并转入盛有水和苯酚/氯仿的管中。在水浴中存放。

8)         每管振荡大约5秒,接着离心2分钟。离心间隙,标记一套新的不含RNA酶的EP管。

9)         把管放回水浴中。取下顶部水层,确保不要取到底层(注:你得浪费靠近表面的约20 μl液体,这对避免污染DNA很重要)。放在水浴中直到做完。

10)     50 μl 不含RNA酶的3M醋酸钠和1 mL乙醇。混合,存放在冰上15分钟。

11)     4°C下离心15分钟。

12)     1 ml 70% 乙醇(不含RNase) 洗涤。4°C下离心10分钟。

13)     尽你所能倒掉乙醇,接着加入400μl 水。放在水浴中约5分钟。

14)     像前面一样用70%的乙醇洗涤,倒置在纸巾上在红外灯下放至干燥。

15)     重新悬浮在50μl的无菌水中(如果原培养体积在5 ml以下用25μl)

16)     放在水浴中5分钟,振荡,离心。

17)     稀释5μlRNA495μl的水中。在A260A240波长下测定吸光度。

 

一些规则

 

1.        总是要戴着手套。

2.        总是要用不含RNA酶的样品管、移液器枪头、塑料容器和溶液。

3.        总是要把RNA储藏液分装到不含RNA酶的容器中,这样你才不会污染到储藏液。

4.        在处理RNA时,或者保持样品管置于冰盒,或者置于超过50°C的水浴中。

 

 

English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


How to cite this protocol: Zheng, W. (2011). RNA Extraction from Yeast. Bio-protocol Bio101: e138. DOI: 10.21769/BioProtoc.138; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook