1.For each probe (control and experimental), set up a separate 100-ml PCR in a 0.5-ml sterile tube, as tabulated below. Either.cDNA inserted in plasmids or genomic DNA can be used as templates for the PCR (see REAGENT SETUP for details on primer design).每个探针(实验组和对照组),在0.5 -ml无菌管设立一个独立的100毫升PCR,正如下面的表。
另外。
互补脱氧核糖核酸插入到基因组DNA质体或可用作模板PCR(见试剂设置有关底漆设计)。
**注意关键：1.很多版本的实验反义核酸探针可以作为一种控制背景染色(见试剂设置)。
然而,我们相信最好的方法来演示特异性是获取相同的空间限制表达模式使用不同的非重叠探测器相同的基因。
2.小心不要污染pcr.使用无菌试管和过滤器的技巧和戴手套3.另外,PCR扩增,cDNAs质粒中可以使用约束线性化酶,独特的站点位于5¢(反义核酸探针)或3¢(对感官探测)来插入。
净化的线性DNA可以通过苯酚/氯仿萃取乙醇沉淀紧随其后。
2| Run the PCR using the conditions tabulated below.使用下面列出的条件运行PCR**暂停点：把扩增好的pcr产品放4℃降温和在-20℃贮藏几个星期。
3| Add the 100-ml PCR to a Microcon YM-50 column and add 400 ml of sterile water. Centrifuge for 15–20 min at 1,000 g at room temperature.加入100毫升PCR到Microcon YM-50列并加入400毫升的无菌水。
在室温下1000g离心15 - 20分钟。
**注意关键：膜应该是干的。
如果没有再离心4|Place the Microcon column into a new microfuge tube (provided in the kit), add 20ml of sterile water, vortex briefly and then turn the Microcon column upside down. Spin for 1 min at 1,000 g at room temperature to recover the DNA.把小层析柱放在一个新的离心管(在这个工具包中提供),增加20毫升的无菌水、短暂离心,然后颠倒层析柱。
自旋1分钟1000 g在室温下恢复了DNA**注意关键：离心的步骤应该快速。
离心机1分钟只是为了避免样本太干。
5|Check the quality, quantity and size of the PCR amplification product by loading 1/20 of the preparation on a 1% (wt/vol) agarose gel in 1 TBE buffer. DNA should appear as a band and not as a smear. The 1/20 of the preparation should contain at least 40 ng of DNA.通过装载1/20的稀释液在1*的TBE buffer缓冲液中的1% (wt/vol)的琼脂糖凝胶检查PCR的扩增产物的质量数量和大小。
DNA产物应该以一个条带而不是以一块污点表现出来。
这份1/20的稀释液至少含有40 ng 的DNA。
6| For each probe, add the components tabulated below to a microfuge tube. Mix and incubate for 2 h at 37 1C.按下面表格给每一份在离心管中的谈增加成分，混合均匀后放在37℃下保存2h.7| Add 2 ml of RNase-free DNase I and 18 ml of sterile water. Mix and incubate for 30 min at 37 1C.添加2毫升的RNase-free DNase I和18毫升的无菌水。
在37℃下混合和孵化30分钟。
8| Stop the reaction by adding 1 ul of sterile 0.5 M EDTA and 9 ul of sterile water.停止反应，通过添加1微升的无菌0.5 M EDTA和9微升无菌水。
9| Place a Sigmaspin post-reaction purification column on top of a microfuge tube. Centrifuge for 15 s at 750g.放一个Sigmaspin反应纯化柱到小离心管，750g离心15 s。
10| Break the base of the column and discard the lid. Spin for 2 min at 750g.破坏柱子的基柱，丢弃盖子，750g 离心旋转2 min11| Place the column on a new microfuge tube. Add the RNA template on top of the resin. Centrifuge for 4 min at 750 g. Discard the column.把柱子放在一个新的离心管上，在树脂上加上RNA模板。
750 g 离心4 min，丢掉柱子。
12| Add 1 ml of sterile EDTA 0.5 M and 9 ml of RNAlater to the sample; this protects the RNA from degradation.加1 ml 无菌的EDTA 0.5 M 和9 ml RNAlater到体系里；这样可以防止RNA降解**注意：由地高辛标记的反义（或正义）的RNA 能在-20℃环境下保存数月。
这个探针合成提供了足够的反义(或正义)RNA执行至少40个左右的不同原杂反应。
13| Visualize 1/20 of the synthesized RNA on 1% (wt/vol)agarose gel in 1 TBE buffer after 30 min of electrophoresis at 230 V. A good probe should appear as one or two discretebands on the gel, not as a smear—which would indicate degradation.设想1/20的合成RNA稀释液在1*的TBE buffer缓冲液中的1% (wt/vol)的琼脂糖凝胶在230 V下经过30 min 的电泳后，好的探针应该以一到两条条带在胶上，而不是一块污点，用来显示降解。
14| Set up pairs of male and female fish in breeding tanks the night before eggs are to be collected. Separate the male and female with a diagonal plastic divider.在鱼卵收集前的晚上建立一个饲养雄鱼和雌鱼的缸，用一个塑料对角分隔器将雄鱼与雌鱼分开饲养。
15| The next morning, when the light goes on, place the upper part of the breeding tank in a clean lower part filled with fresh water. Remove the divider of the breeding tank and let the fish mate for 10–20 min. The eggs laid will sink to the bottom of the breeding tank.第二天天亮后，把饲养缸上半部分移到一个新鲜的淡水体里。
移动分离器，让鱼交配10–20 min。
鱼卵就会沉到水底。
**注意：成人斑马鱼在野外交配时通常在广下。
在养鱼设施中,绝大多数的鱼产卵5 min 到1 h后把灯打开。
可以在上午晚些时候可以获得额外的蛋,但它们通常质量低劣。
16| Collect the eggs from the bottom tank by pouring them into Petri dishes.从水缸里收集沉在水底的鱼卵，放至培养皿里。
**注意：避免一个培养皿中放太多的鱼卵17|Clean the clutch under a stereomicroscope with a diascopic stand; discard dirt and unfertilized eggs using a Pasteur capillary pipette (with a large opening) and pipette pump filler.清理收集鱼卵的培养皿，用消毒过的移液器（有大口）吸掉坏的鱼卵或未受精的鱼卵。
18| Place the embryos in a 100-ml beaker, covered with a minimal amount of water (10 ml).把有用的胚胎放到一个100-ml大烧杯，烧杯里加合适的最少量水10-ml。
19| Pour 3 ml of pronase (1/100 wt/vol, warmed to 28.5 1C) into the beaker and incubate for 1 min. Gentle pronase treatment progressively softens the chorion without damaging the embryos. Alternatively, embryos can be dechorionated by hand using sharpened forceps, but this is a slow process, which can be used only for a small quantity of embryos.倒3毫升链霉蛋白酶(1/100 wt/vol, warmed to 28.5 1C)放入烧杯，孵化1分钟。