搜索

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

下载 PDF 引用 收藏 1 提问与回复 分享您的反馈

本文章节

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.

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.

简介

为了消除由于年龄差异而引起的结果变化,需要在一些测定中使用大的同步的蠕虫群体。 获得同步蠕虫的两种方法包括通过漂白和产蛋制备鸡蛋。 前者通常产生比后者更多的后代,然而,产蛋可以产生比蛋制备更好的同步群体。

材料和试剂

  1. 一般化学品(Sigma-Aldrich)
  2. 常规漂白剂(Clorox)
  3. M9缓冲区
  4. 蠕虫溶解解决方案

设备

  1. Votex(科学工业)
  2. 真空泵(Welch)
  3. 15 ml锥形管
  4. 60×15mm培养皿(VWR)

程序

  1. 通过漂白蛋制备
    1. 块饥饿的蠕虫到种子(与细菌食物,大肠杆菌 OP50-1)NGM板(60×15mm)上。 允许蠕虫在25℃下生长约2天。
    2. 一旦你有很多鸡蛋/成人,倒入6毫升的 M9缓冲液< a>到板上,轻轻地旋转它以移除蠕虫
    3. 将5毫升蠕虫转移到15毫升锥形管中
    4. 向试管中加入5 ml的2x 蠕虫裂解液。 br />
    5. 涡流管最大速度大约4分钟,或直到你看到很少完整的成虫(通常漂白不超过6分钟,否则蛋会被杀死)。
    6. 以〜1,000×g离心1分钟。
    7. 仔细倾析上清液,不要扰乱蠕虫丸
    8. 用10 ml的 M9缓冲液洗涤两次(旋转〜1,000 xg 1分钟)
    9. 添加7 ml的 M9缓冲液,以重悬鸡蛋沉淀。< br />
    10. 让鸡蛋在20℃摇动过夜孵化。 由于没有食物,幼虫应该在L1阶段停止。
    11. 第二天,让管坐在板凳上10 - 15分钟,饥饿的L1蠕虫可以通过重力沉淀。
    12. 除去大部分上清液,并将液体分布到接种板上或液体培养基中 注意:此协议还用于清除蠕虫菌的细菌和酵母菌污染。

  2. 蛋打了
    蚯蚓在20℃下具有7个卵/小时的最大产卵速率。
    1. 挑选10-15个妊娠蠕虫/板
    2. 在所需温度(15,20或25℃)孵育所需时间(2-6小时)
    3. 通过吸盘或挑选来清除成人。
    4. 成长直到后代处于适当的阶段

参考文献

  1. Sulston,J。& Hodgkin,J。(1988)Methods。 在:线虫Caenorhabditis elegans 。 (Ed。):W.B. 木。 Cold Spring Harbor Laboratory Press:New York,587-606
  2. Shapira,M。和Tan,M.W。(2008)。 Caenorhabditis elegans先天免疫的遗传分析 方法Mol Biol 415:429-442。
  • 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: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:He, F. (2011). Synchronization of Worm. Bio-protocol Bio101: e56. DOI: 10.21769/BioProtoc.56;
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。

AR
University of Delaware
1. Is this protocol referenced in any journal? Also, is 20 degree overnight incubation essential or room temperature works?
2/20/2014 1:29:14 PM Reply
Fanglian He
Department of Biology, University of Pennsylvania, USA

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.

2/21/2014 5:45:14 PM