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技術文章

植物基因組DNA 快速提取(50 次)實驗步驟

發布時間:2022-06-14 閱讀:613

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;">植物基因組DNA 快速提取(50 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;">本方法可用于植物細胞DNA 的快速提取,可提取107 細胞,其質量能夠滿足各種分子生物學要求。

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;">組成:
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 用時充分混勻
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 無水/酒精,充分混勻)
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;">步驟:
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. 組織≤0.1g,液氮研磨,加入0.3ml 溶液B,反復混勻,室溫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,充分混勻,室溫放置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 離心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,室溫放置5min,≥8000rpm 離心1min,棄上清。
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,混勻,≥8000rpm 離心1min,棄上清。
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,混勻,≥8000rpm 離心30-60s,棄上清。
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. 重復步驟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,離心30-60s,吸干上清,室溫敞開蓋晾干約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,混勻,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 離心30-60s,取上清,即為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. 組織若多,可適當加大溶液B 和溶液D 的用量,其它成份不變。
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. 混勻一定要溫和,否則DNA 大分子將被打斷,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. 適用新鮮組織、嫩組織,盡可能去除葉脈、葉柄。
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. 液氮研磨一定要保證組織在研磨過程中形如干粉狀。
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. 用戶可依實驗需要,適當調整溶液F 加入量,溶液F 越少,DNA 濃度越高,但產量略有損失,若想大限度提高產量,可重復步驟9、10。兩次所得DNA 可合在一處。

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