农业科学与技术（英文版）
農業科學與技術（英文版）
농업과학여기술（영문판）
AGRICULTURAL SCIENCE & TECHNOLOGY
2010年
3期
41-42,155
,共3页
徐平丽%赵晋平%孟静静%李保龙%李新国%郭峰
徐平麗%趙晉平%孟靜靜%李保龍%李新國%郭峰
서평려%조진평%맹정정%리보룡%리신국%곽봉
拟南芥%PCR%DNA快速提取
擬南芥%PCR%DNA快速提取
의남개%PCR%DNA쾌속제취
Arabidopsis thaliana%PCR%DNA rapid extraction
[目的]介绍一种适于拟南芥PCR检测的DNA快速提取方法.[方法]通过对常规DNA提取方法的改进(省去液氮研磨和苯酚提取的步骤),获得可以快速大批量地提取拟南芥DNA样品的方法.于1.5 ml Eppendorf管内加入400 μl DNA提取液[内含200 mmol/L Tri(pH值7.5),25 mmol/L EDTA(pH值8.0),250 mmol/L NaCl,0.5% SDS(W/V)],剪取拟南芥叶片材料少许(1片以下)加入400 μl DNA提取液,用微型研磨棒将叶片捣碎至溶液成绿色,放置3～5 min;加入400 μl 氯仿/异戊醇(体积比24∶1),混合均匀,12 000 r/min离心5 min;取上清液至另一1.5 ml Eppendorf管内,加入300 μl异丙醇,颠倒混合均匀,室温下放置5 min,12 000 r/min离心5 min;弃去上清液,以70%乙醇漂洗,放置晾干,加入100 μl灭菌超纯水溶解,4 ℃放置备用.以随机抽取的拟南芥转基因株系和突变株系为样品进行验证.[结果]经过琼脂糖凝胶电泳及紫外吸收检测,DNA样品完整且污染少,PCR扩增目的片段结果良好,适于作为PCR反应的模板.经过对随机抽取的拟南芥转基因株系和突变株系的PCR检测,阳性植株目的基因扩增条带清晰,无假阳性,试验结果理想.[结论]该方法适用于拟南芥DNA样品的快速提取、PCR检测及拟南芥突变体筛选工作.
[目的]介紹一種適于擬南芥PCR檢測的DNA快速提取方法.[方法]通過對常規DNA提取方法的改進(省去液氮研磨和苯酚提取的步驟),穫得可以快速大批量地提取擬南芥DNA樣品的方法.于1.5 ml Eppendorf管內加入400 μl DNA提取液[內含200 mmol/L Tri(pH值7.5),25 mmol/L EDTA(pH值8.0),250 mmol/L NaCl,0.5% SDS(W/V)],剪取擬南芥葉片材料少許(1片以下)加入400 μl DNA提取液,用微型研磨棒將葉片擣碎至溶液成綠色,放置3～5 min;加入400 μl 氯倣/異戊醇(體積比24∶1),混閤均勻,12 000 r/min離心5 min;取上清液至另一1.5 ml Eppendorf管內,加入300 μl異丙醇,顛倒混閤均勻,室溫下放置5 min,12 000 r/min離心5 min;棄去上清液,以70%乙醇漂洗,放置晾榦,加入100 μl滅菌超純水溶解,4 ℃放置備用.以隨機抽取的擬南芥轉基因株繫和突變株繫為樣品進行驗證.[結果]經過瓊脂糖凝膠電泳及紫外吸收檢測,DNA樣品完整且汙染少,PCR擴增目的片段結果良好,適于作為PCR反應的模闆.經過對隨機抽取的擬南芥轉基因株繫和突變株繫的PCR檢測,暘性植株目的基因擴增條帶清晰,無假暘性,試驗結果理想.[結論]該方法適用于擬南芥DNA樣品的快速提取、PCR檢測及擬南芥突變體篩選工作.
[목적]개소일충괄우의남개PCR검측적DNA쾌속제취방법.[방법]통과대상규DNA제취방법적개진(성거액담연마화분분제취적보취),획득가이쾌속대비량지제취의남개DNA양품적방법.우1.5 ml Eppendorf관내가입400 μl DNA제취액[내함200 mmol/L Tri(pH치7.5),25 mmol/L EDTA(pH치8.0),250 mmol/L NaCl,0.5% SDS(W/V)],전취의남개협편재료소허(1편이하)가입400 μl DNA제취액,용미형연마봉장협편도쇄지용액성록색,방치3～5 min;가입400 μl 록방/이무순(체적비24∶1),혼합균균,12 000 r/min리심5 min;취상청액지령일1.5 ml Eppendorf관내,가입300 μl이병순,전도혼합균균,실온하방치5 min,12 000 r/min리심5 min;기거상청액,이70%을순표세,방치량간,가입100 μl멸균초순수용해,4 ℃방치비용.이수궤추취적의남개전기인주계화돌변주계위양품진행험증.[결과]경과경지당응효전영급자외흡수검측,DNA양품완정차오염소,PCR확증목적편단결과량호,괄우작위PCR반응적모판.경과대수궤추취적의남개전기인주계화돌변주계적PCR검측,양성식주목적기인확증조대청석,무가양성,시험결과이상.[결론]해방법괄용우의남개DNA양품적쾌속제취、PCR검측급의남개돌변체사선공작.
[Objective] The aim was to introduce a rapid DNA extraction method for PCR detection of Arabidopsis thaliana.[Method] Through the improvement of conventional DNA extraction method,a rapid Arabidopsis thaliana DNA extraction method was obtained.With randomly selected Arabidopsis thaliana transgenic strains and mutants as samples,the method was verified.[Result] After electrophoresis,UV absorption detection,it was found that DNA samples are complete and less pollution,and the result of PCR amplification objective fragment was good which proved DNA is suitable as a template for PCR reaction.After PCR detection,positive plants gene amplified bands were clear,without false-positive,and the test results were satisfactory.[Conclusion] The method is suitable for rapid extraction of Arabidopsis thaliana DNA and PCR detection.
