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    离心机应用一直强调的是什么?

    发布时间:2023-03-28 15:7:55??????点击:

      离心机应用一直强调的是——应根据实验目的和实验需要选择合适的离心机。

      高速,低速,迷你还是大容量,都是要根据自身实验来选择得。

      比如常见的质粒DNA的分离、纯化研究就需要离心机,而且不同的实验方式离心机种类还不一样。

      详细讲解如下:

      一、细菌的培养和收集

      将含有质粒pBS的DH5α菌种接种在LB固体培养基(含50ug/ml Amp)中, 37C培养12-24小时。用无菌牙签挑取单菌落接种到5ml LB液体培养基(含50ug/ml Amp)中,37°C振荡培养约12小时至对数生长后期。

      二、质粒DNA少量快速提取

      质粒DNA小量提取法对于从大星转化子中制备少量部分纯化的质粒DNA十分有用。这些方法共同特点是简便、快速,能同时处理大星试样,所得DNA有一定纯度,可满足限制酶切割、电泳分析的需要。

      (—)、煮沸法:

      1、将1.5ml培养液倒入离心管中,4C下12000 g微量离心机离心30秒。2、弃上清,将管倒置于卫生纸上几分钟,使液体流尽。

      3、将菌体沉淀悬浮于120ml STET溶液中,涡旋混匀。

      4、加入10ml新配制的溶菌酶溶液(10mg/ml),涡旋振荡3秒钟。5、将离心管放入沸水浴中,50秒后立即取出。

      6、用微量离心机4℃下12000g离心10分钟。

      7、用无菌牙签从离心管中去除细菌碎片。8、取20ml进行电泳检查。

      [注意]1.对大肠杆菌可从固体培养基上挑取单个菌落直接进行煮沸法提取质粒DNA.

      ⒉煮沸法中添加溶菌酶有─定限度,浓度高时,细菌裂解效果反而不好。有时不同溶菌酶也能溶菌。

      3.提取的质粒DNA中会含有RNA,但RNA并不干扰进一步实验,如限制性内切酶消化,亚克隆及连接反应等。

      (二)少量DNA纯化系统

      的少量DNA纯化系统可快速有效的抽提质粒DNA,整个过程只需15分钟。提取的质粒可直接用于DNA测序、酶切分析体外转录等。

      该系统中所含试剂和柱子可以用于50次1-3ml质粒培养液的分离和纯化,试剂包括10ml细胞悬浮液,10ml细胞裂解液;10ml中和液,50ml/izard少量DNA纯化树脂,50ml柱洗液(使用前加95%乙醇至120ml )和50支微型柱。

      1、1-3ml过夜培养细胞液4C下12000g台式高数离心机离心1-2分钟。

      2、去除上清液,菌体细胞悬浮于200ul细胞悬浮液中,充分混合,并移入离心管中。3、加200ul细胞裂解液,颠倒离心管数次,直到溶液变清亮。

      4、加200ul中和液,颠倒离心管数次。

      5、4℃下12000g离心5分钟,取上清液于新的离心管中。6、加1ml 少量DNA纯化树脂,颠倒离心管数次以充分混匀。

      7、取一次性注射器,取出注塞,并使注射筒与微型柱连接,用移液枪将上述混合液加入注射筒中;,并用注塞轻推,使混合物进入微型柱。8、将注射器与微型柱分开,取出注塞,再将注射简与微型柱相连;加入2ml柱洗液,并用注塞轻推,使柱洗液进入微型柱。

      9、取出微型柱置于离心管中,离心2分钟以除去微型柱中的柱洗液。

      10、将微型柱放在一个新离心管中,加50pulTE(或水)于微型柱中,静止1分钟后,4℃下12000g台式高数离心机离心20秒。11、丢弃微型柱,将离心管中的质粒DNA贮于4℃或-20°℃冰箱。

      以上就是关于离心机在不同质粒DNA分离、纯化研究中选择不同的讲解,所以在选购离心机的时候,要多跟离心机厂家沟通,购买合适自己的离心机,以免造成资源浪费


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