英文版冻存程序.docVIP

Topic: Freezing C. elegans using liquid freezing solution Equipment and Reagents: ? S Buffer [129 ml 0.05 M K2HPO4, 871 ml 0.05 M KH2PO4, 5.85 g NaCl] ? S Buffer (see above) + 30% glycerin (v/v) (autoclave) ? 1.8 ml cryotube vials Methods: 1. Use one large, 2-3 medium, or 5-6 small NGM plates that have lots of freshly starved L1-L2 animals. Synchronized animals can be obtained by bleaching(alkaline hypochlorite). Wash the plates with 0.6 ml S Buffer for each vial you will freeze, repeat three times. Collect liquid in a sterile test tube. 2. Add an equal volume of S Buffer + 30% glycerin. Mix well. 3. Aliquot 1.0 ml of mixture into 1.8 ml cryovials labeled with strain name and date. 4. Pack the cryovials in a small styrofoam box with slots for holding microtubes or use a commercial styrofoam shipping box. 5. Place the box in a -20°C freezer overnight . 6. Place the box in a -80°C freezer overnight (or for at least 12 hours). 6. The next day transfer the vials to their permanent freezer locations. Thaw one vial as a tester to check how well the worms survived the freezing. Topic: Thawing C. elegans frozen using Liquid Freezing Solution Equipment and Reagents: Methods: 1. Remove a vial from freezer and allow it to thaw at room temperature until all ice has turned to liquid. 2. Pour the contents onto one large NGM plate with E. coli OP50 lawn. You should see worms wiggling after just a few minutes. 3. After 2-3 days, transfer 10-15 animals individually to separate plates. Allow the animals to reproduce for one generation and score the progeny for correct phenotypes. Topic: 线虫同步化 Equipment and Reagents: M9缓冲液：3g KH2PO4 ，6g Na2HPO4 , 5g NaCl ,1ml 1 mol/L MgSO4 加水至1L。 线虫裂解液：0.5 mol/L NaOH , 2.5 %次氯酸。 Methods: （1）首先培养出大量处于良好生理状态的成虫期线虫。 （2）将线虫用M9缓冲液洗脱下来，3000转每分钟离心1分钟，去除上层的缓冲液。 （3）加入1毫升的裂解液，颠倒混匀约3-5分钟。 （4）当观察到虫体已经完全裂解时，3000转每分钟离心1分钟，去除上层裂解液。 （5）用M9缓冲液洗涤3次。 （6）将底层的虫卵用移液枪吸出，撒在涂有OP50的新培养基上。 （7）根据线虫各发育时期的间隔，在特定时间即可得到大量同步化的特定发育时期线虫动物。通常3天后即可获得同步化年轻成虫。 Note: Topic: 线虫的培养