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[Bio101] Expansion of Worms for Microarray, IP, ChIP and Similar Experiments
[Bio101] 用来进行芯片,免疫共沉淀(IP),染色质免疫共沉淀 (ChIP)分析等的蠕虫扩大培养

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Abstract

This protocol describes the basic work-flow of expanding nematode culture under lab conditions, which serves for the subsequent preparation of RNA (microarray), protein (IP), and DNA/protein (ChIP).

Materials and Reagents

  1. Plates and culture materials:
    1. High growth (HG) plates
    2. Normal growth (NG) plates  
    3. OP50 or RNAi bacteria culture
    4. LB medium (autoclaved)
      Note: Prepare enough plates (with extra ones to replace contaminated plates).

  2. Other materials:
    1. Antibiotics (carbenicillin, tetracycline)
    2. IPTG
    3. Cholesterol
    4. CaCl2
    5. MgSO4
    6. KPO4
    7. KOH
    8. Sodium hypochlorite
    9. M9 buffer
    10. NP40 buffer
    11. O/N culture
    12. Triton X-100 (Sigma-Aldrich, catalog number: 9002-93-1 )
    13. Sodium hypochlorite (Thermo Fisher Scientific, catalog number: SS290-1 )
    14. 2x bleach solution (see Recipes)

Equipment

  1. Beckman centrifuge and rotor (Beckman Coulter)
  2. Low-temperature incubator (Thermo Fisher Scientific)
  3. 15-ml conical tubes

Procedure

  1. Before Day 1
    1. Prepare HG plates (10 cm in diameter) and NG plates (15 cm).  Prepare enough medium as described in recipe.  Autoclave, cool down to 60 °C, and then add all the supplements (cholesterol, CaCl2, MgSO4, KPO4, antibiotics, etc). Pour ~15 ml/ 10 cm-plate, ~100 ml/ 15 cm-plate.
      Tip 1. Certain RNAi bacteria do not appear to grow well in the presence of both carbenicillin and tetracycline. In this case, omit tetracycline from the culture and use.

  2. Day 1
    1. Inoculate a single colony of OP50 in 5 ml LB or RNAi bacteria in 5 ml LB (supplemented with 100 μg/ml carbenicillin & 12.5 μg /ml tetracycline) O/N (~16 h), 37 °C, 300 rpm.
      Tip 2. Certain RNAi bacteria do not appear to grow well in the presence of both carbenicillin and tetracycline. In this case, omit tetracycline from the culture and use carbenicillin only to keep the selection pressure. The Tn10 transposon confers tetracycline resistance, which simultaneously knocks out the rnc14 gene that encodes an RNase to enhance the production of dsRNA.
      Tip 3. Prepare a mini-prep of plasmid for the RNAi bacteria and confirm the insert for your gene of interest by PCR (T7 primers) & DNA sequencing (M13 forward primer). The RNAi clones from Julie Ahringer’s lab are based on low copy-number plasmid, so don’t be surprised if the concentration is below detection by spectrophotometry.
    2. Inoculate the O/N bacteria culture in 1 L of LB or LB with antibiotics. Grow O/N (~16 h), 37 °C, 300 rpm.
      Cool the O/N culture on ice (it can be stored in the cold room for a few days). Transfer the culture into centrifuge bottles, spin at 7,000 rpm, 10 min, 4 °C.
      While spinning, make - Make 60 ml NP40 buffer before beginning (30 ml 2x buffer + 6 ml 10% NP40 + 24 ml cold ddH2O - chill on ice!).
      Washing sample in each bottle w/ 250 ml cold H2O. Spin 7,000 rpm 10 min.
      Tip 4. Be extremely careful to avoid any possible contamination to the OP50 culture. Good practice of antiseptic operations is always a must to start your own experiments. Seed a few OP50/NG plates to check contamination. OP50, a uracil auxotroph, should form only a thin layer on NG plate. It is considered to be non-pathogenic to worms. Any weird change of color/smell of your bacteria culture may indicate potential contamination by other types of bacteria.  
    3. Seed OP50 (0.5 ml) on 10-cm HG plates (diameter = 8.5 cm, surface area = 57 cm2). Seed RNA bacteria (1.0 ml) on 15-cm NG /carbenicilin (100 μg/ml)/ IPTG (0.5 mM to 1.0 mM) plates (diameter = 14.0 cm, surface area = 154 cm2).
      Incubate the plates at 37 °C, O/N. Cool down to room temperature before use. Alternatively, put on bench for a few days (adding too much bacteria is not a good idea, since it takes time to dry on the plates).
      Store the plates (seeded with bacteria) in cold room, if not used immediately. Most RNAi bacteria plates can be used within 2 weeks to 1 month (and particularly, for certain potent RNAi clones).
      Tip 5: Typically, 6 to 8 HG plates should provide sufficient numbers of gravid adult animals for hypochlorite synchronization (bleaching) and production of eggs (up to several hundred thousand) for 40 to 50 NG/ carb/ IPTG plates.
      However, different strains could have different development/growth conditions (e.g., mitochondria mutant animals such as clk-1 or isp-1 display slow growth and have less progeny) and optimal conditions should be determined before you start any large-scale experiments.

  3. Day 2
    1. Place two agarose plugs of well-fed animals (e.g., wild-type N2) onto each HG plate.
      Prepare another set on the 2nd day, 3rd day, so on and so forth, to prepare enough biological replicates.

  4. Day 4
    1. When most worms are at d1-d2 stages (it takes ~ 4 to 5 days from starved population), start collection.
    2. Wash worms off from HG plates with M9 containing 0.2% Triton X-100 (0.2 ml per 100 ml). Use glass pipet and 15-ml conical tubes.
    3. Need to wash the plates a few times in order to collect as many gravid animals as possible.
    4. Spin in a bench-top IEC clinical centrifuge at speed 5 (~700 x g), 30 sec.
    5. Remove the supernatant carefully and wash worms w/ M9 buffer, once. Centrifuge again, remove supernatant and leave ~7 ml liquid in the 15-ml conical tube.
    6. Add equal volume of 2x bleach. Agitate/shake the tube slowly yet firmly for ~ 2 to 3 min.
      Tip 6: Optimal incubation time with bleach solution requires a pilot test. The best time to stop is when you see broken carcasses are about to disappear. Remember that you need to collect the eggs by centrifugation for another 30 sec, and during this short period of time, bleach will continue to exert more effects to break the body, and also, causes damage to the eggs. Definitely, avoid over-bleaching!
    7. Start centrifugation for 30 sec to spin down eggs when can still be seen in the tubes. Remove supernatant, and wash immediately with plenty of M9 buffer, at least twice.
    8. Add 10 ml M9 buffer and rock the tube on a rotator at desired temperature (typically, 20 °C), O/N.
      Please refer to Caenorhabditis elegans/Phenotypic assay/Synchronization of worm for further description of hypochlorite synchronization.

  5. Day 5 and beyond
    1. Take a few microliter of M9 and count the numbers of arrested L1 (swimming). Presence of many dead eggs would suggest over-bleaching, which may or may not affect your experiments.
    2. Seed ~6,000 arrest L1 per 15-cm NG/ Carb/ IPTG RNAi plate (or ~2,000 - 2,500 per 10-cm plate).  At least 4,000 animals are required to prepare ≥5 μg of total RNA. You need to concentrate arrested L1 if the density is too low. Otherwise, adding too much M9 buffer on a plate will cause hypoxia.
      Tip 7: Do not add too many animals on a plate. Nematodes love to burrow, and this is one reason why you should not use old plates. One way to solve this problem is to increase the concentration of agar from 2.0% to 3.0%.

  6. Whenever necessary, feed animals with concentrated bacteria that you have prepared before. Start with less arrested L1 (e.g., 3,000 - 5,000) to avoid starvation.
    Tip 7: It takes wild-type animals ~50 - 54 h to come out of arrested L1 and develop to young adult (YA) (w/o eggs). Majority (>90%) of animals should be synchronized as young adults at this point. The time is ~68 - 72 h for daf-2 mutant animals, such as daf-2(e1370), while these animals could never be perfectly synchronized (you will see a mixed population of L3, L4 and YA).
  7. Collect animals at appropriate time points according to your own experiments.
    Tip 8: Compact pellet of ~4,000 YA/d1 adult is ~25 μl in volume. Typically, less than 0.5 ml of worm pellets can be collected from a single 15-cm plate, which can provide enough RNA for at least 200 RT-PCR reactions. We can collect up to several hundred mg of proteins from 10,000 adults.

Recipes

  1. 2x Bleach solution (prepare fresh solution every time before use)
    60 ml sodium hypochlorite
    30 ml 5 N KOH
    10 ml ddH2O

Acknowledgments

This protocol was adapted from work performed by members of the Kenyon lab, including PZ. PZ was supported by a postdoctoral fellowship from the Larry Hillblom Foundation.

References

  1. The Ahringer RNAi library

简介

该协议描述了在实验室条件下扩展线虫培养物的基本工作流程,其用于随后制备RNA(微阵列),蛋白质(IP)和DNA /蛋白质(ChIP)。

材料和试剂

  1. 板材和培养材料:
    1. 高生长(HG)平板
    2. 正常生长(NG)平板
    3. OP50或RNAi细菌培养物
    4. LB培养基(高压灭菌)
      注意:准备足够的板(用额外的板替换污染的板)。

  2. 其他材料:
    1. 抗生素(羧苄青霉素,四环素)
    2. IPTG
    3. 胆固醇
    4. CaCl <2>
    5. MgSO 4 4 /
    6. KPO 4
    7. KOH
    8. 次氯酸钠
    9. M9缓冲区
    10. NP40缓冲区
    11. O/N文化
    12. Triton X-100(Sigma-Aldrich,目录号:9002-93-1)
    13. 次氯酸钠(Thermo Fisher Scientific,目录号:SS290-1)
    14. 2x漂白溶液(参见配方)

设备

  1. Beckman离心机和转子(Beckman Coulter)
  2. 低温培养箱(Thermo Fisher Scientific)
  3. 15毫升锥形管

程序

  1. 在第1天之前
    1. 准备HG板(直径10厘米)和NG板(15厘米)。 准备足够的介质,如食谱中所述。 高压灭菌,冷却至60℃,然后加入所有补充物(胆固醇,CaCl 2,MgSO 4,KPO 4,抗生素, 等等)。 倒入〜15ml/10cm平板,〜100ml/15cm平板 提示1 。某些RNAi细菌在羧苄青霉素和四环素的存在下似乎不生长良好。在这种情况下,从培养物中省略四环素并使用

  2. 第1天
    1. 在37℃,300rpm下,在5ml LB(补充有100μg/ml羧苄青霉素和12.5μg/ml四环素)O/N(约16h)的LB LB或RNAi细菌中接种OP50的单个菌落。
      提示2。某些RNAi细菌在羧苄青霉素和四环素的存在下似乎不会生长良好。在这种情况下,从培养物中省略四环素,并仅使用羧苄青霉素以保持选择压力。 Tn10转座子赋予四环素抗性,其同时敲除编码RNase的rnc14基因以增强dsRNA的产生。
      为RNAi细菌制备质粒的小量制备物,并通过PCR(T7引物)& PCR检测目的基因的插入片段。 DNA测序(M13正向引物)。来自Julie Ahringer实验室的RNAi克隆基于低拷贝数质粒,因此如果浓度低于通过分光光度法检测的浓度,则不要惊讶。
    2. 接种O/N细菌培养物在1 L的LB或LB与抗生素。生长O/N(约16小时),37℃,300转/分 冷却O/N文化在冰上(它可以存储在寒冷的房间几天)。将培养物转移到离心瓶中,以7,000rpm,10℃,4℃旋转 在旋转的同时,在开始之前制备60ml NP40缓冲液(30ml 2x缓冲液+ 6ml 10%NP40 + 24ml冷的冰冷的ddH 2 O冰箱)。
      在每个瓶中洗涤样品w/250ml冷H 2 O。旋转7,000 rpm 10分钟。
      提示4。要特别小心,避免对OP50培养物造成任何可能的污染。良好的消毒操作实践总是必须开始自己的实验。种几个OP50/NG板以检查污染。 OP50,尿嘧啶营养缺陷型,应在NG板上仅形成薄层。它被认为是蠕虫的非致病性。任何细菌培养物的颜色/气味的奇怪变化可能表明其他类型的细菌的潜在污染。  
    3. 在10cm HG板(直径= 8.5cm,表面积= 57cm 2/s)上的种子OP50(0.5ml)。在15cm NG /羧苄青霉素(100μg/ml)/IPTG(0.5mM至1.0mM)板(直径= 14.0cm,表面积= 154cm 2/s)上种植RNA细菌。
      在37℃,O/N孵育平板。使用前冷却至室温。或者,放在台上几天(添加太多的细菌不是一个好主意,因为它需要时间在板上干燥)。
      将板(种细菌)放在冷室,如果不立即使用。大多数RNAi细菌平板可在2周至1个月内使用(特别是对于某些有效的RNAi克隆)。
      提示5:通常,6至8个HG板应提供足够数量的妊娠成年动物用于次氯酸盐同步(漂白)和产生鸡蛋(高达几十万),用于40至50个NG /碳水化合物/IPTG板 然而,不同的菌株可能具有不同的发育/生长条件(例如,线粒体突变动物例如clk-1 或 isp-1 显示缓慢生长并且具有较少的后代),并且在开始任何大规模实验之前应该确定最佳条件

  3. 第2天
    1. 将充分饲养的动物(例如,例如野生型N2)的两个琼脂糖塞置于每个HG板上。
      在第2天,第3天,第2天等等,准备另一组,以此类推,准备足够的生物复制品。

  4. 第4天
    1. 当大多数蠕虫处于d1-d2阶段(需要大约4至5天的饥饿人口)时,开始收集。
    2. 用含有0.2%Triton X-100(0.2ml/100ml)的M9从HG板洗涤蠕虫。 使用玻璃吸管和15毫升锥形管。
    3. 需要洗几次板,以收集尽可能多的妊娠动物
    4. 在台式IEC临床离心机中以速度5(〜700×g/g),30秒旋转。
    5. 小心除去上清液和洗涤蠕虫W/M9缓冲液,一次。再次离心,除去上清液,在15ml锥形管中留约〜7ml液体
    6. 加入等体积的2x漂白剂。搅拌/缓慢摇动管子约2〜3分钟 提示6 :使用漂白溶液的最佳培养时间需要进行中试测试。最好的时候停止是当你看到破碎的屠体即将消失。记住,你需要通过离心收集鸡蛋另外30秒,在这短暂的时间内,漂白将继续发挥更多的影响,以打破身体,还造成对鸡蛋的损害。绝对,避免过度漂白!
    7. 开始离心30秒以旋转蛋,当仍然可以看到在管中。取出上清液,立即用大量M9缓冲液清洗,至少两次。
    8. 加入10ml M9缓冲液,并在旋转器上在所需温度(通常为20℃),O/N下摇动试管。
      有关次氯酸盐同步的进一步说明,请参阅 线虫/表型分析/蠕虫同步 。

  5. 第5天及以后
    1. 取几微升的M9,并计数逮捕的L1(游泳)的数量。 存在许多死蛋会暗示过度漂白,这可能或可能不会影响您的实验
    2. 种子〜每个15cm NG/Carb/IPTG RNAi平板约6000个逮捕L1(或每10cm平板约2,000-2,500个)。 需要至少4,000只动物来制备≥5μg的总RNA。 如果密度太低,你需要集中滞留的L1。 否则,在板上加入太多的M9缓冲液会导致缺氧。
      提示7 :不要在盘子上添加太多的动物。 线虫喜欢挖洞,这是为什么你不应该使用旧板的一个原因。 解决这个问题的一种方法是将琼脂的浓度从2.0%增加到3.0%

  6. 如果需要,喂食动物用你以前准备的浓缩的细菌。从较少逮捕的L1开始(例如,3,000 - 5,000),以避免饥饿 提示7 :野生型动物〜50〜54小时出来的被逮捕的L1和发展成年轻成年人(YA)(W/O鸡蛋)。在这一点上,大多数(> 90%)动物应当作为年轻成年人同步。对于daf-2突变体动物,例如daf-2(e1370),时间是〜68-72小时,而这些动物不可能完全同步(你将看到混合人口L3,L4和YA)。
  7. 根据自己的实验在适当的时间点收集动物。
    提示8 :〜4,000 YA/d1成年人的紧密颗粒体积约为25μl。通常,可以从单个15cm板中收集小于0.5ml的蠕虫小球,其可以为至少200个RT-PCR反应提供足够的RNA。我们可以从10,000个成年人收集高达数百毫克的蛋白质

食谱

  1. 2x漂白溶液(每次使用前准备新鲜溶液)
    60 ml次氯酸钠
    30 ml 5 N KOH
    10ml ddH 2 O 2/

致谢

该协议改编自Kenyon实验室的成员(包括PZ)所进行的工作。 PZ由Larry Hillblom基金会的博士后研究金支持。

参考文献

  1. Ahringer RNAi库
  • English
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引用:Zhang, P. (2011). Expansion of Worms for Microarray, IP, ChIP and Similar Experiments. Bio-protocol Bio101: e129. DOI: 10.21769/BioProtoc.129;
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Yuanqing Lin
Basically, I'm trying to perform RNAi and then qRT-PCR.

I'm a little unclear about the volume of RNAi culture required to seed a 15cm RNAi plate. The protocol said grow up a 1L culture, spin down and then wash in 250mL cold H2O. After that do I resuspend in H2O or the NP40 buffer? If it is NP40 buffer, what is this buffer and what does it do? What is the volume that I should resuspend the culture in?
6/1/2012 1:48:17 PM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

If you refer to the first step of RNAi bacteria prep --- culture a single colony O/N in 1L of LB/Carb 100ug per ml/ Tet 12.5ug per ml (never grow them longer than 14 hours) and then concentrate it by centrifugation. Then wash the RNAi bacteria pellet with LB/Carb/Tet once (it is OK to wash it with sterile H2O), and resuspend the pellet with LB/Carb/Tet (1L to 50ml, 20X concentrated). Store it at 4C and this reservoir will be used as food later. Be careful with potential contamination from bacteria or mold. I have read an article from Michael Petrascheck’s lab about preparing bacteria stock for lifespan assays (JoVE 2011 49 Solis & Petrascheck). They also included anti-fungal in their recipe.

To prepare a 15-cm RNAi plate, you just need to seed ~1.0ml regular RNAi culture (not the concentrated stock). Once you have seeded the worms (less than 6,000 per plate, it’d better to use 2.5% or 3.0% agarose to reduce burrow), remember to feed them with bacteria every other day. Spread ~1.0ml or more concentrated stock onto the plate, and make sure that it will dry soon (you don’t want to add too much liquid onto the surface). I’d say 1 plate of worms is quite enough for qPCR.

BTW, I didn’t use liquid worm culture --- I used solid agar plates instead (you need to be careful with contamination problem, and it’s better to have several extra plates as backup).

6/1/2012 2:19:58 PM


Yuanqing Lin

I am wondering whether your RNAi agar plates contain Tet? And to make sure there is no contamination problem, do you just try to be careful or do you use any anti-fungal stuff?

6/1/2012 2:23:22 PM


Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Our stock agar plates are NG/Carb/IPTG (no Tet at all). Some people in the lab prefer not to use plates that already contain IPTG, and they dilute the bacteria ~5X to 10X and then add IPTG to 1.0mM for induction (either in liquid culture, or mix IPTG and diluted bacteria and then seed on the plate). This RNAi method appears to work pretty well.

In my case, I didn’t include the anti-fungal agent as I was not sure whether it would have any lifespan effects. So try to be careful with your bench and clean it up with 70% ethanol before and after use. Keep the plates closer to the burner. Always seed the plates a few days before you start the worm culture, which would allow you to identify contaminations (typically mold) on the plates. This should work just fine. If you happen to see sporadic mold contamination, you can chunk the agar away and fix the hole with fresh 2% agarose.

6/1/2012 2:33:22 PM