欢迎您, 登录 | 注册

首页 | English

X
加载中

Attaining a large synchronized population of worms is desirable for use in some assays in order to eliminate variation in results due to age differences. Two ways to get synchronized worms include egg preparation via bleaching and egg lay. The former in general yields more progeny than the latter, however, egg lay can generate a better synchronized population than egg preparation.

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] Synchronization of Worm
[Bio101] 蠕虫的同步化

作者: Fanglian He
4/5/2011, 12333 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.56

[Abstract] Attaining a large synchronized population of worms is desirable for use in some assays in order to eliminate variation in results due to age differences. Two ways to get synchronized worms include egg preparation via bleaching and egg lay. The former in general yields more progeny than the latter, however, egg lay can generate a better synchronized population than egg preparation.

[Abstract] 一些实验需要使用大量同时期的蠕虫以消除时期不同带来的结果误差,有两种方法获得同时期的蠕虫,一种是经过漂白的虫卵制备,另一种是产卵。前者比后者有更多的后代,但是产卵可以产生更多同步化的蠕虫。

Materials and Reagents

  1. General chemicals (Sigma-Aldrich)
  2. Regular bleach (Clorox)
  3. M9 buffer
  4. Worm lysis solution

Equipment

  1. Votex (Scientific Industries)
  2. Vacuum pump (Welch)
  3. 15 ml conical tube
  4. 60 x 15 mm petri dish (VWR)

Procedure

  1. Egg preparation via bleaching
    1. Chunk starved worms onto a seeded (with bacterial food, E coli OP50-1) NGM plate (60 x 15 mm). Allow the worms to grow for about 2 days at 25 °C.
    2. Once you have plenty of eggs/adults, pour 6 ml of M9 buffer onto the plate and gently swirl it to dislodge the worms.
    3. Transfer 5 ml worms to a 15 ml conical tube.
    4. Add 5 ml of 2x worm lysis solution to the tube.
    5. Vortex the tube at max speed for approximately 4 min or until you see very few intact adult worms (usually bleach no longer than 6 min, otherwise the eggs will be killed).
    6. Centrifuge at ~1,000 x g for 1 min.
    7. Carefully decant the supernatant without disturbing the worm pellet.
    8. Wash twice with 10 ml of M9 buffer (spin at ~1,000 x g for 1 min)
    9. Add 7 ml of M9 buffer to resuspend the egg pellet.
    10. Let eggs shake overnight at 20 °C to hatch. Since there is no food the larvae should be halted at the L1 stage.
    11. Next day, let the tube sit on the bench for 10 - 15 min and starved L1 worms can precipitate by gravity.
    12. Remove most of supernatant and distribute the liquid onto seeded plates or into liquid culture.
      Note: This protocol is also used to remove bacterial and yeast contamination from a worm strain.

  2. Egg lay
    Gravid worms have a maximal egg-laying rate of 7 eggs/h at 20 °C.
    1. Pick 10-15 gravid worms/plate.
    2. Incubate at desired temperature (15, 20, or 25 °C) for desired time (2-6 h).
    3. Remove adults by suction off the plate or picking.
    4. Grow until progeny are at appropriate stage.

References

  1. Sulston, J. & Hodgkin, J. (1988) Methods. In: The Nematode Caenorhabditis elegans. (Ed.): W.B. Wood. Cold Spring Harbor Laboratory Press: New York, 587-606.
  2. Shapira, M. and Tan, M. W. (2008). Genetic analysis of Caenorhabditis elegans innate immunity. Methods Mol Biol 415: 429-442.

材料与试剂

 

1.        一般的化学试剂 (Sigma)

2.        Regular bleach (Clorox)

3.        60x15 MM petri dish (VWR)

 

仪器

 

1.        Votex (Scientific Industries)

2.        真空泵 (Welch)

 

步骤

 

1.        通过漂白虫卵制备

1)       大量饥饿的蠕虫放置在种子(细菌食物,E coli OP50-1)的NGM板(60x15 MM)上, 25oC下生长2天。

2)       等有大量的卵/成虫,加入6 mL of M9 缓冲液到板上,并轻轻的摇晃到以取出蠕虫。

3)       转移5 ml 蠕虫到15mL 离心管中。

4)       5 ml of 2x 蠕虫裂解液到管中。

5)       高速震荡管约4分钟或直到你看见少量的完整的成年蠕虫(通常漂白不超过6min,否则将杀死虫卵)。

6)       ~1,000 x g离心1 minute

7)       倒掉上清

8)       10mL M9 缓冲液洗两次 (~1,000 x g 离心1 minute )

9)       加入7mL M9缓冲液重悬卵沉淀

10)   20oC过夜孵化。由于没有食物给幼虫,因此幼虫会停止在L1阶段。

11)   次日,将离心管静置1015 min。饥饿的L1阶段的蠕虫通过重力沉淀。

12)   去除上清,将液体加入到种子板或液体培养基中。

注:这个方法也可以用于去除蠕虫的细菌或酵母污染。

2.        产卵

妊娠蠕虫

妊娠蠕虫在20oC有一个最大的产卵率,7个卵/小时

10-15个沉淀的蠕虫/

1)       152025孵育2-6h

2)       用吸管去除成虫。

3)       在适当阶段直到后代长大。

 

参考文献

 

1.        Sulston, J. & Hodgkin, J. (1988) Methods. In: The Nematode Caenorhabditis elegans. (Ed.): W.B. Wood. Cold Spring Harbor Laboratory Press: New York587-606.

2.        Shapira M., Tan M.W. (2008). Genetic analysis of Caenorhabditis elegans innate immunity. Methods in Molecular Biology 415: 429-42. 


 

 

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: He, F. (2011). Synchronization of Worm. Bio-protocol Bio101: e56. DOI: 10.21769/BioProtoc.56; 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
2/20/2014 1:29:14 PM  

AR
University of Delaware

1. Is this protocol referenced in any journal? Also, is 20 degree overnight incubation essential or room temperature works?

2/21/2014 5:45:14 PM  

Fanglian He (Author)
Carnegie Institution for Science at Stanford

Hi,
This protocol was adapted from Man-Wah Tan's group (see reference 2 above). 20 degree overnight incubation is not essential. Room temperature should work too.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register