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      技术文章

      植物基因组DNA 快速提?。?0 次)实验步骤

      发布时间:2022-06-14 阅读:96

      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">植物基因组DNA 快速提?。?0 次)
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">概述:
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">本方法可用于植物细胞DNA 的快速提取,可提取107 细胞,其质量能够满足各种分子生物学要求。

      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">组成:
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">1.溶液A 1.2ml RNase A 10mg/ml。-20℃保存
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">2.溶液B 60ml
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">3.溶液C 180ml
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">4.溶液D 3ml Resin 用时充分混匀
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">5. 溶液E 50ml 洗液(用前加入75ml 无水/酒精,充分混匀)
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">6.溶液F 25ml TE buffer

      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">步骤:
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">1. 组织≤0.1g,液氮研磨,加入0.3ml 溶液B,反复混匀,室温5min。
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">2. 加入0.8ml 溶液C,溶液A 20μl,充分混匀,室温放置20min。
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">3. ≥8000rpm 离心3min。
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">4. 取上清,加入50ul 溶液D,室温放置5min,≥8000rpm 离心1min,弃上清。
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">5. 加入800μl 溶液C,混匀,≥8000rpm 离心1min,弃上清。
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">6. 加入1ml 溶液E,混匀,≥8000rpm 离心30-60s,弃上清。
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">7. 重复步骤6。
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">8. ≥12000rpm,离心30-60s,吸干上清,室温敞开盖晾干约5-10min。
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">9. 取溶液F 200-300μl,混匀,40-50℃水浴3-5min。
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">10. ≥12000rpm 离心30-60s,取上清,即为DNA。

      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">注:
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">1. 组织若多,可适当加大溶液B 和溶液D 的用量,其它成份不变。
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">2. 混匀一定要温和,否则DNA 大分子将被打断,而部分降解。
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">3. 适用新鲜组织、嫩组织,尽可能去除叶脉、叶柄。
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">4. 液氮研磨一定要保证组织在研磨过程中形如干粉状。
      portant;="" white-space:="" orphans:="" 2;="" widows:="" background-color:="" rgb(255,="" 255,="" 255);="" font-variant-ligatures:="" font-variant-caps:="" -webkit-text-stroke-width:="" text-decoration-style:="" initial;="" text-decoration-color:="" initial;"="" style="white-space: normal; caret-color: rgb(51, 51, 51); color: rgb(51, 51, 51); font-family: "Microsoft YaHei", Arial, Helvetica, sans-serif;">5. 用户可依实验需要,适当调整溶液F 加入量,溶液F 越少,DNA 浓度越高,但产量略有损失,若想大限度提高产量,可重复步骤9、10。两次所得DNA 可合在一处。

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