EASYspin 植物RNA快速提取試劑盒操作方法及步驟說(shuō)明書(shū)

1、杭州昊鑫生物科技股份有限公司 htpp:/EASYspin Plant RNA KitEASYspin植物RNA快速提取試劑盒目錄號(hào)：RN09v 試劑盒組成、儲(chǔ)存、穩(wěn)定性：試劑盒組成保存50次(RN0902)裂解液RLT室溫50 ml去蛋白液RW1室溫40 ml漂洗液RW室溫10 ml第一次使用前按說(shuō)明加指定量乙醇RNase-free H2O室溫10 mlPLANTaid室溫5 mlRNase-free吸附柱RA和收集管室溫50套本試劑盒在室溫儲(chǔ)存12個(gè)月不影響使用效果。儲(chǔ)存事項(xiàng)：1. 不合適的儲(chǔ)存于低溫（4或者20）會(huì)造成溶液沉淀，影響使用效果，因此運(yùn)輸和儲(chǔ)存均在室溫下（1525）進(jìn)行。2.

2、 避免試劑長(zhǎng)時(shí)間暴露于空氣中產(chǎn)生揮發(fā)、氧化、PH值變化，各溶液使用后應(yīng)及時(shí)蓋緊蓋子。v 注意事項(xiàng)1. 所有的離心步驟均在室溫完成，使用轉(zhuǎn)速可以達(dá)到13,000 rpm的傳統(tǒng)臺(tái)式離心機(jī)。2. 需要自備乙醇，研缽(可選)。3. 裂解液RLT和去蛋白液RW1中含有刺激性化合物，操作時(shí)戴乳膠手套，避免沾染皮膚，眼睛和衣服。若沾染皮膚、眼睛時(shí)，要用大量清水或者生理鹽水沖洗。4. 關(guān)于DNA 的微量殘留：一般說(shuō)來(lái)任何總RNA提取試劑在提取過(guò)程中無(wú)法完全避免DNA的微量殘留，本公司的EASYspin系列RNA提取產(chǎn)品，由于采取了本公司獨(dú)特的緩沖體系和選擇了特殊吸附能力的吸附膜已經(jīng)清除了絕大部分的DNA殘留，

3、在大多數(shù)RT-PCR 擴(kuò)增過(guò)程中極其微量的DNA殘留影響不是很大，如果要進(jìn)行嚴(yán)格的mRNA表達(dá)量分析如熒光定量PCR，我們建議在進(jìn)行模板和引物的選擇時(shí)：1) 選用跨內(nèi)含子的引物，以穿過(guò)mRNA中的連接區(qū)，這樣DNA就不能作為模板參與擴(kuò)增反應(yīng)。2) 選擇基因組DNA和cDNA上擴(kuò)增的產(chǎn)物大小不一樣的引物對(duì)。3) 將RNA提取物用RNase-free的DNase I 處理。本試劑盒還可以用于DNase I處理后的RNA清潔(cleanup)，請(qǐng)聯(lián)系我們索取具體操作說(shuō)明書(shū)。4) 在步驟去蛋白液RW1漂洗前，直接在吸附柱RA上進(jìn)行DNase I柱上消化處理。購(gòu)買DNA酶柱上消化試劑盒(貨號(hào)：RN34)

4、前可先索取具體操作說(shuō)明書(shū)。5. 關(guān)于復(fù)雜植物樣品提取殘留較多DNA的情況有兩個(gè)選項(xiàng)：1) 在步驟去蛋白液RW1漂洗前，直接在吸附柱RA上進(jìn)行DNase I柱上消化處理。購(gòu)買DNA酶柱上消化試劑盒(貨號(hào)：RN34)前可先索取具體操作說(shuō)明書(shū)。2) 部分較復(fù)雜的植物樣品提取時(shí)，可能殘留較多DNA，可以嘗試本公司的RN38 EASYspin Plus植物RNA提取試劑盒。RN38在RN09 EASYspin植物RNA提取試劑盒基礎(chǔ)上，又獨(dú)家研發(fā)成功基因組DNA清除柱技術(shù)可以有效清除DNA殘留，在大多數(shù)情況下，可以將DNA殘留清除到紫外下觀測(cè)不可見(jiàn)。6. 關(guān)于特別復(fù)雜難提取植物樣品提取失敗或者產(chǎn)量低的情

5、況：一些特別復(fù)雜的植物樣品提取，如葡萄果實(shí)，藍(lán)靛果果實(shí)等，RN09 裂解液RLT無(wú)法提取，需選擇RN53 EASYspin Plus多糖多酚/復(fù)雜植物RNA提取試劑盒。一些樣品產(chǎn)量較低，也可以嘗試RN53 EASYspin Plus多糖多酚/復(fù)雜植物RNA提取試劑盒。RN53配有強(qiáng)力裂解液CLB選項(xiàng)，在很多情況下，可以提取復(fù)雜樣品或者明顯提高產(chǎn)量（詳情請(qǐng)看RN53說(shuō)明書(shū)）。v 操作步驟：（實(shí)驗(yàn)前請(qǐng)先閱讀注意事項(xiàng)）提示： 第一次使用前請(qǐng)先在漂洗液RW瓶加入指定量無(wú)水乙醇!1. 直接研磨法（推薦）:a. 新鮮植物組織稱重后取100mg迅速剪成小塊放入研缽（冰凍保存或者液氮保存樣品可直接稱重后取10

6、0mg放入研缽）， 加入10體積（1ml）RLT和1體積（100l） PLANTaid 室溫下充分研磨成勻漿，注意應(yīng)該迅速研磨讓組織和裂解液RLT立刻充分接觸以抑制RNA酶活性。注：PLANTaid是提取多糖多酚次級(jí)代謝產(chǎn)物色素含量豐富的困難樣品不可缺少成分。b. 將裂解物轉(zhuǎn)入離心管，劇烈搖晃振蕩15秒，13,000rpm離心5-10分鐘，沉淀不能裂解的碎片和結(jié)合有多糖多酚的PLANTaid。c. 取480l裂解物上清（在不超過(guò)RNA吸附柱能力的情況下可以取更多或者全部上清，這樣可以提高產(chǎn)量）轉(zhuǎn)到一個(gè)新離心管。加入上清體積一半的無(wú)水乙醇(0.5體積)，此時(shí)可能出現(xiàn)沉淀，但是不影響提取過(guò)程，立即

7、吹打混勻，不要離心。d. 立刻接操作步驟的步驟3。2. 液氮研磨法（提取復(fù)雜，易降解樣品時(shí)推薦此法）:a. 取500l裂解液RLT，轉(zhuǎn)入1.5ml離心管中，加入50l PLANTaid混勻備用。b. 液氮中研磨適量植物組織成細(xì)粉后，取50mg細(xì)粉轉(zhuǎn)入上述裝有RLT和PLANTaid的離心管，立即用手劇烈振蕩20秒，充分裂解。c. 用吸頭吹打混勻幫助裂解或者劇烈渦旋震蕩直到得到滿意勻漿結(jié)果(或者電動(dòng)勻漿30秒)，可以剪切DNA，降低粘稠度和提高產(chǎn)量。d. 將裂解物13,000 rpm離心5-10分鐘，沉淀不能裂解的碎片和結(jié)合有多糖多酚的PLANTaid。e. 取裂解物上清（在不超過(guò)RNA吸附柱能

8、力的情況下可以取更多的上清，這樣可以提高產(chǎn)量）轉(zhuǎn)到一個(gè)新離心管。加入上清體積一半的無(wú)水乙醇(0.5體積)，此時(shí)可能出現(xiàn)沉淀，但是不影響提取過(guò)程，立即吹打混勻，不要離心。f. 立刻接操作步驟的步驟3。注意：以上液氮研磨法用戶可以根據(jù)需要加倍處理，可以提高產(chǎn)量。也就是使用1ml的裂解液RLT和100l PLANTaid和100mg的樣品。3. 將混合物(每次小于720l，多可以分兩次加入)加入一個(gè)吸附柱RA中，（吸附柱放入收集管中）13,000 rpm離心2分鐘，棄掉廢液。確保離心后液體全部濾過(guò)去，膜上沒(méi)有殘留，如有必要，可以加大離心力和離心時(shí)間。4. 加700l 去蛋白液RW1，室溫放置1分鐘，

9、13,000rpm 離心30秒，棄掉廢液。5. 加入500l漂洗液RW（請(qǐng)先檢查是否已加入無(wú)水乙醇!），13,000 rpm 離心30秒，棄掉廢液。加入500l漂洗液RW，重復(fù)一遍。6. 將吸附柱RA放回空收集管中，13,000 rpm離心2分鐘，盡量除去漂洗液，以免漂洗液中殘留乙醇抑制下游反應(yīng)。7. 取出吸附柱RA，放入一個(gè)RNase free離心管中，根據(jù)預(yù)期RNA產(chǎn)量在吸附膜的中間部位加30-50l RNase free water（事先在70-90水浴中加熱可提高產(chǎn)量）， 室溫放置1分鐘，12,000 rpm 離心1分鐘。8. 如果預(yù)期RNA產(chǎn)量30g，加30-50l RNase fr

10、ee water重復(fù)步驟7，合并兩次洗液，或者使用第一次的洗脫液加回到吸附柱重復(fù)步驟一遍(如果需要RNA濃度高)。洗脫兩遍的RNA洗脫液RNA濃度高，分兩次洗脫合并洗脫液的RNA產(chǎn)量比前者高1530%，但是濃度要低，用戶根據(jù)需要選擇。附錄1：DNA酶柱上消化（詳細(xì)請(qǐng)參考RN34 DNase I 柱上消化試劑盒說(shuō)明書(shū))1. 按照前面所列RN09試劑盒操作步驟操作，直到做完操作步驟3。2. 取45l DNase I buffer和5l RNase free DNase I在離心管輕輕吹打混勻成工作液（處理多個(gè)離心柱子要按照比例放大制備工作液）。3. 向吸附柱RA 中加入350l去蛋白液RW1，12

11、,000 rpm 離心30 秒，棄廢液，將吸附柱放回收集管中。4. 向吸附柱RA 中央加入50l的DNase I 工作液，室溫（20-30）放置15 分鐘。注意直接將工作液滴在膜中央上向膜四周浸潤(rùn)充分和膜接觸，不要讓工作液滴在O型墊圈或是離心柱管壁上掛壁或者掛在墊圈上不能充分和膜接觸。5. 向吸附柱RA 中加入350l去蛋白液RW1， 12,000 rpm 離心30-60 秒，棄廢液，將吸附柱放回收集管中。6. 接操作步驟5完成后續(xù)步驟。附錄2：使用EASYspin/EASYspin Plus系列植物RNA快速提取試劑盒發(fā)表的部分文章100多篇：1. 桃果實(shí)、花、根、葉：Isolation,

12、characterisation and phylogenetic analysis of resistance gene analogues in a wild species of peach (Prunus kansuensis).Canadian Journal of Plant Science, 2011, 91(6): 961-970 2. 櫻桃花、葉、顎等各部位：Over-expression of the PaAP1 gene from sweet cherry (Prunus avium L.) causes early floweri.Journal of Plant Ph

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38、ogical characters of chrysanthemum YUTAI. Plant Cell Tiss Organ Cult 2014, DOI 10.1007/s11240-014-0541-147. 蕎麥和擬南芥：Ectopic expression of FaesAP3, a Fagopyrum esculentum (Polygonaceae) AP3 orthologous gene rescues stamen development in an Arabidopsis ap3 mutant. Gene 2014, 550(2): 20020648. 油松：Differ

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