EASYspinPlus多糖多酚复杂植物RNA快速提取试剂盒操作方式和步骤说明书

1、EASYspin Plus Complex Plant RNA Kit EASYspin Plus多糖多酚/复杂植物RNA快速提取试剂盒 目录号：RN53适用范围：适用于快速提取植物组织细胞总RXA,利用独有基因组DXA清除柱技术可有效清除 电泳可见gD'A残留，RNA可用于反转录PCR,荧光定量PCR等。试剂盒组成、贮存、稳固性：试剂盒组成保存50 次(RN53O1)裂解液CLB室温50 ml裂解液RLT Plus室温25 ml去蛋白液RW1室温40 ml漂洗液RW室温10 ml第一次使用前按说明加指定量乙醇HjO室温10 ml基因组DNA清除柱和收集管室温50套RNase-free

2、吸附柱RA和收集管室温50套木试剂盒在室温贮存12个月不阻碍利用成效。贮存事项：1 .不适合的贮存于低温(4或-20C)会造成溶液沉淀，阻碍利用成效，因此运输 和贮存均在室温下(15C 25C)进行。2 .幸免试剂长时刻暴露于空气中产生挥发、氧化、PH值转变,各溶液利用后应及时 盖紧盖子。注意事项1 . 所有的离心步骤均可在室温完成（4。离心也能够），利用转速能够达到13, 000 rpm 的传统台式离心机，如Eppendorf 5115C或类似离心机°2 . 需要自备B-筑基乙萌，乙醇,研钵（可选）。3 .样品处置量绝对不要超过基因组清除柱DA和和RNA吸附柱RA处置能力，不然造成

3、 DNA残留或产量降低。开始试探实验条件时，若是不清楚样品DNA/RNA含量时可利 用较少的样品处置量，以后依照样品实验情形增加或减少处置量。4 .裂解液CLB和RLT Plus和去蛋白液RW1中含有刺激性化合物，操作时戴乳胶手套， 幸免沾染皮肤，眼睛和衣服.假设沾染皮肤、眼睛时，要用大量清水或生理盐水 冲洗.5 .关于DNA的微量残留：一样说来任何总RNA提取试剂在提取进程中无法完全幸免DNA的微型残留（DNase 消化也无法做到100%无残留），本公司的EASYspin Plus RNA提取产品，由于采 取广本公司独特的缓冲体系和基因组DNA消除柱技术，绝大多数DNA已经被清除， 不需要D

4、Nase消化,可直接用于反转录PCR和荧光定量PCR个别特殊情形如DNA 含型过于丰硕造成残留或要进行严格的mRNA表达坦分析荧光定量PCR,咱们建议 在进行模板和引物的选择时：1）选用跨内含子的引物，以穿过mRNA中的连接区，如此DNA就不能作为模板参与扩 增反映。2） 选择基因组DNA和cDNA上扩增的产物大小不一样的引物对。3） 将RNA提取物用RNase-free的DXase I处置。木试剂盒还能够用于DXase I处置后的RNA清洁（cleanup）,请联系咱们索取具体操作说明书°4）在步骤去蛋白液RW1漂洗前，直接在吸附柱RA上进行DXase I柱上消化处置。购买 DNA

5、陶柱上消化试剂盒（货号：RN34）前可先索取具体操作说明书。操作步骤：（实验前请先阅读注意事项）提示：第一次利用前请先在漂洗液RW瓶加入指定量无水乙静！取1ml裂解液CLB至离心管内（若是CLB有析出或沉淀需先宣于65° C水浴从头 溶解），在裂解液CLB中加入5% B-航基乙醉（lmlCLB加50Hl 0-筑基乙醇）。 倒置混匀后65° C水浴中预热。多个样品依照比例放大侦备。1 .直接研磨法（实验室无液氮情形下或柔软易研磨植物样品推荐此法）：a.新鲜植物组织或冰冻保留样品称重后取100-200mg （水分少的样品如叶片种 子等可加100-150mg,水分多的样品如草售西

6、瓜果实可多加一些）迅速剪成小块 放入研钵，加入1nli CLB （已加有B-貌基乙醇）室温下充分研磨成匀浆，注意 应该迅速研磨让组织和裂解液CLB立刻充分接触以抑制RXA酎活性。P磷基乙醇是裂解液CLB的关键成份，必要的时候能够提高终浓度到10-20%。若是专门复杂植物，能够尝试在裂解液中加入PVP40至终浓度2%.b.将裂解物转入离心管，当即猛烈振荡15秒，短时放回65° C水浴中（5-10 min）,中间偶然倒置1-2次帮忙裂解c 13, OOOrpm离心10分钟，沉淀不能裂解 的碎片。c.取裂解物上清（在不超过基因组DNA消除柱能力的情形下能够取更多的上清,如此能够提高产量）转

7、到一个新离心管。加入卜.清体积一半的无水乙醉体积）,现在可能显现沉淀，可是不阻碍提取进程，当即吹打混匀，不要离心。假设上清表面有漂浮物，用吸头挑开吸取下面液体即可.d.立刻接操作步骤的步骤3。2 .液氨研脚法（适用普遍，提取曳杂难破碎，易降解样品时推荐此法）：a.液凝中研磨新鲜或-70° C冷冻的材料至细粉cb.转移100-200mg细粉（水分少的样品如叶片种子等可加100-150mg,水分多 的样品如草徒西瓜果实可多加一些）加至侦热的裂解液CLB （已加有端基乙 醉）离心管中。当即剧烈涡旋30-60秒或用吸头吹打混匀裂解直取得中意匀浆结 果（或电动匀浆30秒），能够剪切DNA,降低

8、粘制度和提高产量。c.短时放回650 c水浴中（5-10 min）,中间偶然倒置1-2次帮忙裂解,d.将裂解物13, 000 rpm离心10分钟，沉淀不能裂解的碎片。e.取裂解物上消（在不超过基因组DNA消除柱能力的情形下能够取更多的上清, 如此能够提高产量转到一个新离心管。加入上清体积一半的无水乙静体积）. 现在可能显现沉淀，可是不阻碍提取进程，当即吹打混匀，不要离心。假设上清表面有漂浮物，用吸头挑开吸取下面液体即可.f.立刻接操作步骤的步骤3。3 . 将混合物（每次小于720 Mb多能够分两次加入）加入一个基因组清除柱中，（清 除柱放入搜集管中）13, 000 rpm离心2分钟，弃掉废液。

9、确保惠心后液体全数流过去，膜上没有残留，如有必要，能够加大离心力和离心 时刻。4 .将基因组D'A清除柱子放在一个干净2ml离心管内（不用RNAsefree或DEPC处置，一样干净的新离心管即可。也可利用RNA吸附柱配套的新的干净搜集管），在基因组清除柱内加500 u 1裂解液RLT Plus, 13, 000 rpm离心30秒，搜集滤 液（RNA在滤液中），用微量移液器较精准估量漉过液体积（一样为450-500 u 1 左右，港过时候损失体积应该减去），加入倍体积的无水乙醇，现在可能显现沉 淀，可是不阻碍提取进程，当即吹打混匀，不要离心。5 . 立刻将混合物（每次小于720 M 1,

10、多能够分两次加入）加入一个吸附柱RA中，（吸 附柱放入搜集管中）13, 000 rpm离心2分钟，弃掉废液。确保离心后液体全数流过去，膜上没有残留，如有必要，能够加大离心力和离心 时刻.6 .加700U1去蛋白液RW1,室温放置1分钟，13,000rpm离心30秒，弃掉废液。7 . 加入500 m 漂洗液RW （请光检查是不是已加入无水乙醇！），13,000 rpm离心 30秒，弃掉废液。加入500 U 1漂洗液RW,重豆一遍。8 . 将吸附柱RA放回空搜集管中，13,000 rpm离心2分钟，尽可能除去漂洗液，以 避免漂洗液中残留乙醇抑制下游反映。9 .掏出吸附柱RA,放入一个RNase f

11、ree离心管中，依照预期RNA产量:在吸附膜的 中间部位加30-50 P 1 RNase free water （事前在70-90C水浴中加热可提高产 量），室温放置1分钟，12, 000 rpm离心1分钟。10 .若是侦期心A产量30u g,加30-50 Ul RNase free water重夏步骤9,归并两 次洗液，或利用第一次的洗脱液加回到吸附柱重豆步骤一遍（若是需要RNA浓度 而。洗脱两遍的RNA洗脱液浓度高，分两次洗脱归并洗脱液的RNA产量比前者高15 - 30%,可是浓度要低，用户依照需要选择。附录:h DNA酶柱上消化(详细请参考心34 DNase I柱上消化试剂盒说明书)1

12、.依照前面所列RN53试剂盒操作步喋操作，直到做完操作步骤5。2 . 取45 u 1 DNase I buffer和5 u 1 RNase free DNase I在离心管轻轻吹打混匀成 匚作液(处置多个离心柱子要依照比例放大制备工作液3 .向吸附柱RA中加入350U1去蛋白液RW1, 12, 000 rpm离心30秒，弃废液，将 吸附柱放回搜集管中。4 .向吸附柱RA中央加入50U1的DNase I工作液，室温(2(TC-30)放置15分 钟c注意直接将工作液滴在膜中央上向膜周围浸涧充分和膜接触，不要让工作液 滴在0型垫圈或是离心柱管壁上挂壁或挂在垫圈上不能充分和膜接触。5 .向吸附柱RA中

13、加入350 U1去蛋白液RW1, 12. 000 rpm离心30-60秒，弃废液， 将吸附柱放回搜集管中。6 .接操作步骤7完成后续步骤。附录2： RNA含量少样品或RNA复杂产量低的解决方案能够提高样品处置里到300-500mg/2ml裂解液CLB,上清过两根基因组DNA清除柱子， 洗脱下来的RNA,能够两个归并到一根RNA吸附柱上，能够大大提高RNA浓度,附录3：利用EASYspm/EASYspin Plus植物RNA提取系列试剂盒发表文章：100多篇：1. 帙果实、花、根、叶：Isolation, characterisation and phylogenetic analysis of

14、 resistance gene analogues in a wild species of peach (Prunus kansuensis). Canadian Journal ofPlant Science,2020, 91(6): %1-5702. 樱桃花、叶、颗等各部位：Over-expression of the PaAPl gene from sweet cherry (Prunus avium L.) causes early of Plant Physiology, 2021, Available online 1 December 20213. 洋慈根、茎、鱼、叶、嬉推彩

15、等各部位：Cloning and Expression Analysis of A Putative B Class MAD5-box Gene of AcPI in Onion. Scientia Agricultura Sinica, 2021, 15(23) :4759-47694. 芜 著 ： Isolation and Functional Characterisation of the Genes Encoding A S-SphingolipidDesaturase from Brassica rapa. Journal of Genetics andGenomics Volum

16、e 39, Issue 1, January 2021, Pages 47 - 595. 芜 1 ： EXPRESSION, DIVERGENCE AND SOLUTION OF THE CALEOSIN GENE FAMILY IN BRASSICA RAPA. Arch. Biol. Sci.» Belgrade, 65(3), 363776, 2021 D0I:ABS1303S63H6. 番茄叶：Effect of Low Tesiperatiire Stress on the Expression of ProDH Gene and the Activities of the

17、 Proline Dehydrogenase in Leaves of Tosato Seedling. Chinese Agricultural Science Bulletin 2021, 28(10):132-1357. 桅子叶： Isolation of High Quality Total RXA froa Gardenia jaszninoides Agricultural Science , 2S(27):191-19S8. 油桐 果实： Cui Qinain, Han Xiaojiao, Chen Yicun, Zhan Zhiyone> Lin Liyuan, Vang

18、 Yangdong.Isolation and Expression Characteristics of Biotin Carboxyl CarrierProtein Coding Gene (VfBCCP) from Vernicia SILVAE SINICAE. 2021, 48(8): Available online August9. 油桐果实 1 ： Selection of Reliable Reference Genes for Gene Expression Studies Using Real-Time PCR in Tung Tree during Seed Devel

19、opment. PLoS ONE, 2021, 7(8): e130S 110. 紫菜：Molecular cloning and expression analysis of ribosomal protein S7 gene fromPorphyrahaitanensis. JOURNAL OF FISHERIES OF CHIXA, 2020, 35 (12> ： 1S1I-1S2111. 石料：Molecular characterisation of a mitogen-activated protein kinase gene DcMPKl in Dendrobium off

20、icinale. Acta Pharzaceutica Sinica, 2021, 17 (12): 1703-170912. 午i斛 1： ESTs Analysis Reveals Putative Genes Involved in Symbiotic Seed Germination in Dendrobium officinale. Symbiotic Germination Genes in D. officinale. August 2021 Voluzne S Issue S ©7270513. 大豆：RNA-seQ Analysis Reveals Ethylene

21、-Mediated Reproductive Organ Development and Abscission in Soybean (Glycine max L. Merr. ). Plant Mol Biol Rep, 2021, published online: 4 Dec, 202111. 大豆 1： Construction of ethylene regulatory network based on the phytohormones related gene transcriptoze profiling and prediction of transcription fac

22、tor activities in soybean. Acta Physiol Plant, 2021, published online: 12 Dec, 202115. 红花玉 2： Expression Analysis of MAwuAG in Different Organs and Developmental Stages of Magnolia wufeagensis. Chinese Bulletin of Botany, 2021, IS (2): 1 - 516. 毛桃：Cloning and Phylogeny Analysis of PpAP2 Floral Hoool

23、ogous Genes in Peach. Chinese Agricultural Science Bulletin, 2021, 29(7): 99-10117. 五倍子：Cloning and characterisation of a phenylalanine azmonia-lyase gene from Rhus chinensis. Plant Cell Rep, 2021, published online： 15 March, 202118. :五倍子 1： Cloning, charac t er i: at ion and expression of chaicone

24、synthase from zedicinal plant Rhus . Plant Biochem. Biotechnol. DOI sl-919. 青杆 ：cDNA Cloning and Sioinformatic Analysis of the sP?al Gene form Picea wilsonii. Plant Science Journal, 2021, 30(40): 391-10120. 青杆 1： cDXA Cloning and Sioinformatic Analysis of PsbO Gene free Picea Science Research,2021,

25、16(3): 201-20621. 青杆 2： Cloning and Tissue Expression Analysis of PwPSAF in Picea vilsonii. SCIENTIA SILVAS SINICAE. Vol. 49. No. 10, Oct. 2021.22. 洋葱：Molecular Cloning and Transcriptional Analysis of the Putative AGAMOUS Hoeolog AcAG in Onion (Allium cepa. Plant Xol 5iol Rep, DOI s7-y23. 木瓜：XsFAD-

26、gene encodes the enz>>se responsible for the high linoleic acid content in oil accumulated in Xanthoceras sorbifolia seeds. JOURNAL ARTICLE. 2021-6-17.24. 木瓜 1： Tvo novel diacylglycerol acyltransferase genes from Xanthoceras 2 sorbifolia are responsible for its seed oil content. GENH-3S6SS; No

27、. of pages: 9: IC:25. 柑桔：Efficient auto-excision of a selectable marker gene froa transgenic citrus by combining the Cre/loxP system and ipt selection. Plant Cell Rep, DOI s00299013-l1170-x26. 柑桔 1： Expression Analysis of Three Phloem-specific Promoters in Transgenic Poncirus trifoliata. Acta Hortic

28、ulturae Sinica. 2021, 41(1): 1 - S.27. 柑桔 2： Activation of three pathogen-inducible promoters in transgenic citrus (Citrus sinensis Osbeck) after Xanthomonas a>:onopodis pv. citri infection and wounding. Plant Cell Tiss Organ Cult. DOI s3-y.28. 茶梅花藩：Cozaparison and Analysis of Methods of Extracti

29、ng Total RNA from Petals of Camellia sasanQua. Chinese Agricultural Science , 29(28):129-133.29. 桅子：Isolation of High Quality Total RNA frocGardenia jasminoides Eills. Chinese Agricultural Science Bulletin. 2021, 2S(27):19H19S30. 丹参：Genome-wide analysis and molecular dissection of the SPL gene famil

30、y in Salviamiltiorrhiza. 2021 Jan:56(1):3S-5O. doi: . Epub 2021 Nov 20.31. 牡丹：Transcriptome Comparison Reveals Key Candidate Genes Responsible for the Unusual Reblocming Trait in Tree Peonies. Genes Responsible for Reblocming in Tree Peonies. November 2021 Volume S Issue 11e799%32. 东南景天：Role of sulf

31、ur assimilation pathway in cadmium h5-peraccumulation by Sedum alfredii Hance. Ecotoxicology and Environmental Safety. , February 2021, Pages 159 165.33. 山苍子：Identification of appropriate reference genes for normalizing transcript expression by quantitative real tise PCR in Litsea cubeba. TECHNICAL

32、NOTE. Mol Genet Genomics（2021） 288:727 - 737, DOI §00133-013-0785-134. 木本植物：Heterologous gene silencing induced by tobacco rattle virus （TRV） is efficient for pursuing functional genooiics studies in woody plants. ORIGINAL PAPER. Plant Cell Tiss Organ Cult, DOI 53r35. 棉花：Analysis of sea-island

33、cotton and upland cotton in response to Verticillium dahliae infection by RNA sequencing. Sun et al. BMC Genomics 2021, 14:S52 /14712161/11/852.36. 桃子：Biochemical changes and defence responses during the development of peach gumzosis caused by Lasiodiplodia theobromae. Eur J Plant Pathol （2021） 133:

34、193 - 207, DOI s2l.37. 桃子 1 ： Carbohydrate metabolism changes in Prunus persica giumDosis infected with Lasiodiplodia theobrosiae. Phytopathology "First Look" paper 01-13"0025 posted 11/27/2021.38. 海关 The Malus crabapple transcription factor McMYBlO regulatesanthocyanin biosynthesis d

35、uring petal coloration. Scientia Horticultural 166 （2021） 12 - 19.39. 海藻：A rapid and sensitive ziethod for field detection of Prorocentrua donghaiense using reverse transcription-coupled loop-mediated isothermal amplification. Harmful Algae 29（2021） 31 39.10 .油茶：Establish a cDNA"AFLP Technology

36、 System in Camellia oleifera. Molecular PlantBreeding, 2021, , , 611-616.11 . 亚洲百合：Transcriptomic analysis of Asiatic lily in the process of vernalization via RNA-seq. Mol Biol Rep. DOI sOY.12. 毛泡桐：Dynamic egression of novel and conserved microRNAs and their targets in diploid and tetrapioid of Paul

37、ownia tcmentosa. Biochimie xxx (2021) lelO.13. 人参：Cloning and Sequence Analysis Squalene Epoxidase Gene in Panax gin-seng. Journal of Jilin Agricultural University 2021, 36(2): 149-152,1714. 竹莲：Cloning and Sequence Analysis of rbcs Gene from Sasussured involucrdta Kar. et Kir. Chinese Agricultural S

38、cience Bulletin 2021, 30(15): 261-26715. 柑桔 3： Secreted Expression of Cecropin B Gene Enhances Resistance to XanthoiDonas a>:onopodis pv. citri in Transgenic Citrus sinensis ,Tarocco, Acta Horticulturae Sinica 2021, 11(3): 417 - 428 ac. cn16. 菊花：Stem apex detoxification culture markedly improved

39、severalphysiological characters of chrysanthezzrom 'YUTAI，. Plant Cell Tiss Organ Cult 2021. DOI sl-l47.养麦和拟南芥：Ectopic expression of FaesAP3, a Fagopyrum esculentum (Polygonaceae) AP3 orthologous gene rescues stamen development in an Arabidopsis ap3 mutant. Gene 2021, 550(2): 200 - 20618. 油松：Dif

40、ferential expression of SLOW WALKERS hosologue in ovules of fezale sterile mutant and fertile clone of Pinus tabulaeformis. Russian Journal of Developmental Biology 2021, 45(2): 78-8449. 玫瑰花：Precise spatiotemporal modulation of ACC synthase by MPK6 cascade mediates theresponse of rose flowers to reh

41、ydration. The Plant Journal 2021, 79(6): 911 - 95050. 棉花和拟南芥：Functional characterization of GhAKTl, a novel Shaker-like K* channel gene involved in K+ uptake from cotton (Gossypium hirsutiim). Gene 2021, 515(1): 61 - 7151. 棉花和拟南芥 1 ： Upland Cotton Gene GhFPFl Confers Promotion of Flowering Tice and Shade-Avoidance Responses in Arabidop