| 查看: 2491 | 回复: 15 | ||||
[交流]
植物DNA提取
|
||||
|
植物叶片提取基因组DNA，蛋白很多，大家都是用什么方法去除的？我是用一半体积的Tris平衡酚，和等体积的氯仿异戊醇，抽提2次。但是蛋白很多 [ 来自科研家族 OMICS家族 ] |
回复此楼» 收录本帖的淘帖专辑推荐
 科研资源 |
» 猜你喜欢
为呼吸打开通道：依伐卡托的科学之旅
已经有1人回复
-
Dordaviprone（ONC201）：小分子药物在中线胶质瘤中的应用
已经有0人回复
-
化学工程及工业化学论文润色/翻译怎么收费?
已经有260人回复
-
依喜替康：新型喜树碱衍生物的研究进展
已经有1人回复
-
膀胱癌靶向治疗新选择：厄达替尼作用机制与耐药研究进展
已经有0人回复
-
从水母到实验室：腔肠素的发现历程与生物发光奥秘
已经有1人回复
-
线粒体氧化磷酸化的新靶点：S-Gboxin的发现与研究进展
已经有0人回复
-
PROTAC药物开发选择VHL配体：MDK-7526以87%出口向量占有率成首选
已经有0人回复
-
MCC950：NLRP3炎症小体特异性抑制剂的科研应用与前景
已经有0人回复
-
康替唑胺研发17年：如何解决多重耐药菌感染难题
已经有0人回复
» 本主题相关价值贴推荐，对您同样有帮助:
- DNA提取时用到的酚是什么酚？怎么配置？
已经有22人回复
- 动植物样品的采集及DNA样品的提取(ZT)
已经有10人回复
- 植物干样DNA提取试剂盒
已经有15人回复
- 如果想重复利用提取植物样品DNA的小塑料研磨棒，如何去除DNA？
已经有16人回复
- 植物DNA提取问题
已经有8人回复
- 大家都用什么方法提取植物DNA
已经有19人回复
- 植物基因组DNA提取的一些疑问~
已经有10人回复
- 超低温保存植物组织ＤＮＡ提取问题
已经有7人回复
- 【求助/交流】异硫氰酸胍法提取植物RNA中DNA残留问题~回帖就送币！！！
已经有14人回复
- 【求助/交流】提取的植物基因组DNA电泳图，疑问？？？
已经有17人回复
» 抢金币啦！回帖就可以得到:
-
祝大家学业有成，工作顺利，生意兴隆
+1/937
-
江西理工大学2026年博士研究生招生报名公告（第二批次），可以联系马胜灿老师
+2/932
-
【帮转急招】深大计算机/电子信息博士 王牌专业，大牛团队！
+5/765
-
中科院深圳先进技术研究院集成电路先进封装博士后招聘
+1/278
-
复旦大学化学系董安钢-李同涛团队招聘博士后（3-5名）
+3/156
-
征结婚对象，男找女，坐标【北京】【北京】【北京】
+5/155
-
诚邀津门师者共研
+1/76
-
【通知】北京信息科技大学仪器科学与光电工程学院招收博士研究生（2026）
+2/46
-
海南大学国家高层次人才团队硕、博士招生
+1/37
-
【急招】“双一流”高校-新能源材料课题组招收2026年秋季入学博士生1名(湘潭大学)
+1/36
-
双一流大学湘潭大学“化工过程模拟与强化”国家地方联合工程研究中心招收博士生
+1/34
-
安徽农业大学招聘师资博士后以及人才引进教师
+1/24
-
招聘青年教师（有编制）——南京邮电大学柔性电子全国重点实验室徐申课题组
+1/23
-
紧急招收2026年秋季入学博士生1名（湘潭大学 固体废弃物低碳利用湖南省工程研究中心）
+1/12
-
【博士招生】武汉科技大学招收材料、化工、环境、冶金类2026年“申请-考核”制博士生
+1/12
-
QQQ跌破700後資金依然回流，這個位置其實沒那麼弱。
+1/10
-
材料分析测试
+1/8
-
深圳理工大学核酸药物递送与干细胞工程课题组科研助理教授和博士后招聘启事
+1/3
-
【有偿访谈招募】高才通来港后，你过得还好吗？
+1/1
-
一文看懂DNA损伤检测：守护生命蓝图的“基因体检”
+1/1
13楼2012-08-06 13:23:15
★ ★ ★ ★
xiaohaiyu(金币+1): 谢谢参与
西瓜: 金币+3, 鼓励交流 2012-05-20 18:46:11
xiaohaiyu(金币+1): 谢谢参与
西瓜: 金币+3, 鼓励交流 2012-05-20 18:46:11
|
小心的提取会成功的，给你实验步骤 Plant Genomic DNA Extraction using CTAB Introduction The search for a more efficient means of extracting DNA of both higher quality and yield has lead to the development of a variety of protocols, however the fundamentals of DNA extraction remains the same. DNA must be purified from cellular material in a manner that prevents degradation. Because of this, even crude extraction procedures can still be adopted to prepare a sufficient amount of DNA to allow for multiple end uses. DNA extraction from plant tissue can vary depending on the material used. Essentially any mechanical means of breaking down the cell wall and membranes to allow access to nuclear material, without its degradation is required. For this, usually an initial grinding stage with liquid nitrogen is employed to break down cell wall material and allow access to DNA while harmful cellular enzymes and chemicals remain inactivated. Once the tissue has been sufficiently ground, it can then be resuspended in a suitable buffer, such as CTAB. In order to purify DNA, insoluble particulates are removed through centrifugation while soluble proteins and other material are separated through mixing with chloroform and centrifugation. DNA must then be precipitated from the aqueous phase and washed thoroughly to remove contaminating salts. The purified DNA is then resuspended and stored in TE buffer or sterile distilled water. This method has been shown to give intact genomic DNA from plant tissue. To check the quality of the extracted DNA, a sample is run on an agarose gel, stained with ethidium bromide, and visualised under UV light. Materials CTAB buffer Microfuge tubes Mortar and Pestle Liquid Nitrogen Microfuge Absolute Ethanol (ice cold) 70 % Ethanol (ice cold) 7.5 M Ammonium Acetate 55o C water bath Chloroform : Iso Amyl Alcohol (24:1) Water (sterile) Agarose 6x Loading Buffer 1x TBE solution Agarose gel electrophoresis system Ethidium Bromide solution CTAB buffer 100ml 2.0 g CTAB (Hexadecyl trimethyl-ammonium bromide) 10.0 ml 1 M Tris pH 8.0 4.0 ml 0.5 M EDTA pH 8.0 (EthylenediaminetetraAcetic acid Di-sodium salt) 28.0 ml 5 M NaCl 40.0 ml H2O 1 g PVP 40 (polyvinyl pyrrolidone (vinylpyrrolidine homopolymer) Mw 40,000) Adjust all to pH 5.0 with HCL and make up to 100 ml with H2O. 1 M Tris pH 8.0 Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust pH to 8.0 by adding 42 ml of concentrated HCL. Allow the solution to cool to room temperature before making the final adjustments to the pH. Adjust the volume to 1 L with H2O. Sterilize using an autoclave. 5x TBE buffer 54 g Tris base 27.5 g boric acid 20 ml of 0.5M EDTA (pH 8.0) Make up to 1L with water. To make a 0.5x working solution, do a 1:10 dilution of the concentrated stock. 1% Agarose gel 1 g Agarose dissolved in 100 ml TBE Procedure - Grind 200 mg of plant tissue to a fine paste in approximately 500 μl of CTAB buffer. - Transfer CTAB/plant extract mixture to a microfuge tube. - Incubate the CTAB/plant extract mixture for about 15 min at 55o C in a recirculating water bath. - After incubation, spin the CTAB/plant extract mixture at 12000 g for 5 min to spin down cell debris. Transfer the supernatant to clean microfuge tubes. - To each tube add 250 μl of Chloroform : Iso Amyl Alcohol (24:1) and mix the solution by inversion. After mixing, spin the tubes at 13000 rpm for 1 min. - Transfer the upper aqueous phase only (contains the DNA) to a clean microfuge tube. - To each tube add 50 μl of 7.5 M Ammonium Acetate followed by 500 μl of ice cold absolute ethanol. - Invert the tubes slowly several times to precipitate the DNA. Generally the DNA can be seen to precipitate out of solution. Alternatively the tubes can be placed for 1 hr at -20 o C after the addition of ethanol to precipitate the DNA. - Following precipitation, the DNA can be pipetted off by slowly rotating/spinning a tip in the cold solution. The precipitated DNA sticks to the pipette and is visible as a clear thick precipitate. To wash the DNA, transfer the precipitate into a microfuge tube containing 500 μl of ice cold 70 % ethanol and slowly invert the tube. Repeat. ((alternatively the precipitate can be isolated by spinning the tube at 13000 rpm for a minute to form a pellet. Remove the supernatant and wash the DNA pellet by adding two changes of ice cold 70 % ethanol )). - After the wash, spin the DNA into a pellet by centrifuging at 13000 rpm for 1 min. Remove all the supernatant and allow the DNA pellet to dry (approximately 15 min). Do not allow the DNA to over dry or it will be hard to re-dissolve. - Resuspend the DNA in sterile DNase free water (approximately 50-400 μl H2O; the amount of water needed to dissolve the DNA can vary, depending on how much is isolated). RNaseA (10 μg/ml) can be added to the water prior to dissolving the DNA to remove any RNA in the preparation (10 μl RNaseA in 10ml H2O). - After resuspension, the DNA is incubated at 65o C for 20 min to destroy any DNases that may be present and store at 4o C. - Agarose gel electrophoresis of the DNA will show the integrity of the DNA, while spectrophotometry will give an indication of the concentration and cleanliness. DNA quality confirmation – Prepare a 1 % solution of agarose by melting 1 g of agarose in 100 mL of 0.5x TBE buffer in a microwave for approximately 2 min. Allow to cool for a couple of minutes then add 2.5 μl of ethidium bromide, stir to mix. – Cast a gel using a supplied tray and comb. Allow the gel to set for a minimum of 20 min at room temperature on a flat surface. – Load the following into separate wells o 10 μL 1kb ladder o 5 μL sample + 5 μL water + 2 μL 6x Loading Buffer – Run the gel for 30 min at 100 V – Expose the gel to UV light and photograph (demonstration) – Confirm DNA quality, presence of a highly resolved high molecular weight band indicates good quality DNA, presence of a smeared band indicates DNA degredation. |
» 本帖附件资源列表
-
欢迎监督和反馈：小木虫仅提供交流平台，不对该内容负责。
本内容由用户自主发布，如果其内容涉及到知识产权问题，其责任在于用户本人，如对版权有异议，请联系邮箱：xiaomuchong@tal.com - 附件 1 : Plant Genomic DNA Extraction by CTAB _2__Fiona.pdf
2012-05-20 15:33:10, 95.39 K
8楼2012-05-20 15:33:14
3楼2012-05-18 22:42:59
4楼2012-05-19 08:24:34
5楼2012-05-19 08:26:22
6楼2012-05-19 11:08:04
7楼2012-05-20 13:57:10
9楼2012-05-20 15:51:39
10楼2012-05-20 16:44:24
12楼2012-05-21 08:19:37
14楼2013-06-10 11:32:45
15楼2013-09-19 10:32:46
16楼2015-02-12 23:43:37
简单回复
2012-05-18 21:36
回复
xiaohaiyu(金币+1): 谢谢参与
hmily861911楼
2012-05-20 17:32
回复
xiaohaiyu(金币+1): 谢谢参与