搜索

Spot Assay for Yeast
酵母斑点实验   

下载 PDF 引用 收藏 3 提问与回复 分享您的反馈

本文章节

Abstract

This protocol can be used to compare the cell growth rate of yeast under different growth conditions. It involves the serial dilution and spotting of yeast colonies.

Materials and Reagents

  1. Yeast cells
  2. YES medium

Equipment

  1. Multichannel Pipetman (Eppendorf)
  2. OmniTray (V&P Scientific)
  3. Microfuge tube
  4. Standard laboratory spectrophotometer

Procedure

  1. Start cultures from a 2 day old plate. Use pipette tip to pick up strains and resuspend them in 1.5 ml YES medium or water.
  2. Vortex and transfer 1 ml to another microfuge tube. Test OD600 for an accurate reading, the OD should be between 0.1 and 0.5.
  3. Dilute the rest of the suspension to 16 OD600, around 1.6 x 106 cells per ml, 1,600 cells per μl. That is 4,800 or 5,000 cells per 3 μl. Spot 3 μl cells on each position.
  4. If using OmniTray, start with the 1st column (8 wells in each column).
  5. Do 5 fold serial dilution from the 1st to 5th column. Leave the 6th column empty. Transfer another 8 strains culture into the 7th column and do another 5 fold dilution.


Acknowledgments

This protocol has been modified and adapted in the Espenshade Lab, Johns Hopkins School of Medicine. Funding to support different projects that have used this protocol has come from NIH – National Heart, Lung, and Blood Institute, National Institute of Allergy and Infectious Diseases, the Pancreatic Cancer Action Network, and the American Heart Association.

References

  1. Tong, Z., Gao, X. D., Howell, A. S., Bose, I., Lew, D. J. and Bi, E. (2007). Adjacent positioning of cellular structures enabled by a Cdc42 GTPase-activating protein-mediated zone of inhibition. J Cell Biol 179(7): 1375-1384.

简介

该方案可用于比较不同生长条件下酵母的细胞生长速率。 它涉及酵母菌落的连续稀释和点样。

材料和试剂

  1. 酵母细胞
  2. 是中等

设备

  1. 多通道移液器(Eppendorf)
  2. OmniTray(V& P Scientific)
  3. 微杯管
  4. 标准实验室分光光度计

程序

  1. 从2天龄的平板开始培养。 使用移液器吸头提取菌株,并重悬在1.5毫升是介质或水。
  2. 涡旋和转移1毫升到另一个微量离心管。 测试OD <600>以获得准确的读数,OD应在0.1和0.5之间
  3. 将悬浮液的其余部分稀释至16OD 600,约1.6×10 6个细胞/ml,1,600个细胞/μl。这是4,800或5,000细胞每3微升。点在每个位置3微升细胞
  4. 如果使用OmniTray,从第1列开始(每列8个孔)。
  5. 从第1列至第5列进行5倍连续稀释。将第6列留空。转移另外8株培养物到第7列,做另一个5倍稀释

致谢

该协议已经在约翰霍普金斯医学院的Espenshade实验室中修改和改编。资助支持不同的项目,使用这个协议来自NIH - 国家心脏,肺和血液研究所,国家过敏和传染病研究所,胰腺癌行动网络和美国心脏协会。

参考文献

  1. Tong,Z.,Gao,X.D.,Howell,A.S.,Bose,I.,Lew,D.J.and Bi,E。(2007)。 通过Cdc42GTPase激活蛋白介导的抑制区实现的细胞结构的相邻定位。 a J Cell Biol 179(7):1375-1384。
  • 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。
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tong, Z. (2012). Spot Assay for Yeast. Bio-protocol 2(1): e16. DOI: 10.21769/BioProtoc.16.
提问与回复

(提问前,请先登录)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。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。

Bhavna Veer
Pondicherry University, India
Hi
Please let me know the procedure to make YPDA plates with the compound. I`m actually unable to validate my results with Spot Assay. I`ve used the concentration with which I got some sensitive mutants in 96 well liquid system. In fact, I have used a little higher concentration as well in the solid media. But the yeast mutant strains which have shown sensitivity to the drug in 96 well liquid system did not show any sensitivity in the plate system. Treated plate was as good as the control plate after the incubation.
Please help me with this.
Thank you.
5/8/2014 11:57:26 PM Reply
How I can explin my reselt if I use 25 ,28 ,30 ,31 ,34 ,36 c ?
I need explin the reselt plaese?
12/4/2012 12:18:06 AM Reply
Zongtian Tong
Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, USA

Sorry, I am not sure what do you ask. The 96 plate picture is just for reference. You can use any plate you like.

12/22/2012 9:49:56 PM


What is the easiest way to determine if a colony is yeast? Is there a simple test kit that you know of?

Thank you
Chandra
3/21/2012 12:04:02 AM Reply
Zongtian Tong
Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, USA

You can determine if the colony is yeast or bacteria by looking at the color and shape of the colony. Bacteria colony is usually yellowish and the shape is smooth.

You can also use a pipette tip to touch the colony, drop it into water and transfer some to a microscope slide. Observe the colony under light microscope. Bacteria is much smaller than yeast. E.Coli is rod shape. Budding yeast has buds.

3/22/2012 12:54:46 PM


Muhammad Sameeullah
Institute of Plant Science and Resources

How to concentrate yeast culture from OD600=0.70 to 1.0? please suggest easiest way. thanks.

9/16/2013 6:08:17 PM