一、实验目的:快速提取动物组织中的总RNA(18S,28S;离心吸附柱不吸附5S)
二、实验器材及试剂:研磨仪,离心管,移液枪,电泳槽凝胶成像等;无水乙醇,液氮,琼脂糖。
三、实验材料:鸡肉组织。
四、实验步骤
1)鸡肉组织
2)鲜组织用解剖刀迅速切成小碎离心管中,加入小号研磨珠,将管子盖严后放入液氮中冷冻,设置参数,点击运行即可开始研磨。
3)取适量组织细粉转入装有组织裂解液RLT的离心管中,用手剧烈振荡若干秒,充分裂解。用带钝针头的一次性注射器抽打裂解物若干次或直到得到满意匀浆结果(或者电动匀浆若干秒),可以剪切DNA,降低粘稠度和提高产量。
4)将匀浆后裂解物离心,沉淀可能存在的裂解困难的碎片或者不溶物,将裂解物上清小心转到一个新离心管。
5)接操作步骤项下3。
6)较精确估计裂解物(上清)体积,加入等体积的乙醇(请先检查是否已加入无水乙醇!),此时可能出现沉淀,但是不影响提取过程,立即吹打混匀,不要离心。
7)立刻将混合物加入一个吸附柱RA中,(吸附柱放入收集管中)离心60秒,弃掉废液。
8)加去蛋白液RW1,室温放置几秒,离心30秒,弃掉废液。
9)如果DNA残留明显,可在加入RW1后室温放置几分钟再离心。
10)加入漂洗液RW(请先检查是否已加入无水乙醇!),离心30秒,弃掉废液。加入漂洗液RW,重复一遍。
11)将吸附柱RA放回空收集管中离心2分钟,尽量除去漂洗液,以免漂洗液中残留乙醇抑制下游反应。
12)取出吸附柱RA,放入一个RNase free离心管中,根据预期RNA产量在吸附膜的中间部位加30-50μl RNase free water,室温放置1分钟,离心1分钟。
13)如果预期RNA产量>30μg,加30-50μl RNase free water重复步骤8,合并两次洗脱液,或者使用第一次的洗脱液加回到吸附柱重复步骤一遍(如果需要RNA浓度高)。
14)洗脱两遍的RNA洗脱液浓度高,分两次洗脱合并洗脱液的RNA产量比前者高15–30%,但是浓度要低,用户根据需要选择。
五、实验结果:提取的鸡肉组织RNA点样量为1ul,鸡肉组织RNA两个条带亮度相当,提示出现降解。
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