Protocol 实验方法聚合平台
EverLab正在努力的检索，请稍候^_^1 0 - 0 篇 | 共 0 篇
免责声明及举报方式
本站注册用户不得侵犯包括他人及科研机构著作权在内的知识产权和其他合法权利，请勿在未经授权的情况下录入任何涉及侵权的实验方法。除非由您个人原创或经合法授权后转载。任何由用户发布的相关内容而引起的知识产权纠纷或其他法律纠纷，其责任均在用户本人，与本站无关。用户提交共享实验方法即表示用户本人已确认该方法不违反国家相关法律法规及本站协议，并且已拥有该方法的合法、完整的版权或者合法、完整的修改及转载授权。
涉嫌侵权实验方法举报方式
本站在提供给大家一个共享科研实验方法平台的同时，也完全尊重并保护原创者及机构的合法知识产权。但在工作运营中难免会有疏漏之处，所以大家一旦发现有涉嫌侵权的实验方法，我们官方支持并欢迎各位积极举报投诉，让本平台能在合法公正的基础上，切实为大家的科研工作带来便利。本站举报投诉格式如下：
涉嫌侵权实验方法举报：
1. 被举报实验方法链接：（请填写涉嫌侵权实验方法浏览页的URL链接地址）
2. 方法被举报投诉原因：（请填写被举报方法侵权的详细原因或相关有效证明）
以上信息您可通过以下联系方式提交给我们，网站编辑部会在接到举报后两个工作日内核实查证，并在一周内妥善处理。谢谢大家对本站的大力支持和信任！
电子邮箱：
电话号码： 010-
QQ 号 码：
北京库巴扎信息科技有限公司发一个自己的半内源性免疫共沉protocol(免疫,抗体,质粒,孵育) - 免疫实验 - 生物秀
标题: 发一个自己的半内源性免疫共沉protocol(免疫,抗体,质粒,孵育)
摘要: [发一个自己的半内源性免疫共沉protocol(免疫,抗体,质粒,孵育)] 最近才做成功的试验，拿出来和战友们分享经验。这个试验曾经困扰我很久，终于大功告成了。希望能给战友们一点帮助。祝大家试验顺利。其实，免疫共沉一般只分内源和外源。外源就是两个蛋白都是外源性高表达的。而内源只要一个蛋白为细胞本底即为内源。我这个protocol介绍的方法，诱饵蛋白是高表达的，目标蛋白是细胞本底的，为和两个蛋白都是内源的区别，就暂称为半内源性吧。整个实验大致分为两部分：细胞转染和抗生素(a 关键词:[抗体 质粒 免疫 孵育 蛋白 脂质体 上清]……
最近才做成功的试验，拿出来和朋友们分享经验。这个试验曾经困扰我很久，终于大功告成了。希望能给朋友们一点帮助。祝大家试验顺利。其实，免疫共沉一般只分内源和外源。外源就是两个蛋白都是外源性高表达的。而内源只要一个蛋白为细胞本底即为内源。我这个protocol介绍的方法，诱饵蛋白是高表达的，目标蛋白是细胞本底的，为和两个蛋白都是内源的区别，就暂称为半内源性吧。整个实验大致分为两部分：细胞转染和抗生素(antibiotic)压制，免疫共沉一，细胞转染：实验材料：1。目标细胞（以Hela为例）2。完全培养基（10%血清），无血清培养基3。10cm dish（corning公司的）4。1ml和200ml枪头，EP管（灭菌）5。PBS（灭菌）6。各种量程的枪7。目标质粒（带有细胞筛选抗性，以puro最好）8。细胞抗生素(antibiotic)（以puro为例）9。Lipinfectin 2000（Invitrogen公司）二，免疫共沉1。RIPA细胞裂解液（买的国产碧云天的，一定要选“弱”的那个）2。cocktail蛋白酶抑制剂(protease inhibitor)（罗氏公司的，EDTA-free，一个小药片）3。Protein G（PIERCE公司的试剂盒）4。Primary Antibody（Santa Cruz公司polyclone）5。Secondary Antibody（国产二抗，Ap标记）6。BSA（进口分装Amersco）7。细胞刮8。TBS Buffer9。低温离心机10。低温摇床Protocol：Day 1。准备好目标质粒24ug，纯度为转染级。（一般来说，对于普通细胞系，国产小提试剂盒即可，我用的是博大泰克的。所有牌子里最滥的一个。）将细胞铺到10cm dish 里，以Hela来说，大约是5X106，各种细胞不同，一般为下午接，第2天早上大约60~70%丰度。Day 2。所有细胞用溶液，平衡到室温。取两个EP管，分别加入500ml无血清培养基，先取其中一个，稀释24ug质粒，孵育5分钟，再取另一个Ep管，稀释40ul脂质体。将质粒溶液逐滴滴入脂质体溶液，小心混合，孵育20分钟。在这时候，取出细胞培养板，用PBS小心地洗细胞2~3次再加入约5ml无血清培养基到10cm dish。孵育结束后，将混合有质粒和脂质体的溶液，逐滴加入到平皿里，小心晃动平皿，使之均匀。6小时后，弃去培养基，换上完全培养基继续培养。Day 3。24小时后（从加入质粒和脂质体混合溶液的时间开始算），加入嘌呤霉素2ug/ml。继续培养24小时。Day 4。配制cocktail蛋白酶抑制剂(protease inhibitor)储液。一片溶于1.5ml无菌水中即可，为7X母液。但是做共沉的话，浓度要高一点。冰预冷PBS。取出平皿，吸去培养基 ，用冰预冷的PBS洗细胞2次，加入400ulRIPA细胞裂解液，再加入85ul的cocktail溶液。用细胞刮刮取细胞，反复多次，直至肉眼看不见白色的细胞层。所有操作冰上进行。在低温离心机里，以12’000rpm离心20分钟。取上清。先留出20ul作为western阳性对照。其余的加入一抗3ug。放入低温摇床150rpm，4度摇过夜。Day 5。吸取30ulProtein G ，50%浓度，加入PBS 500ul，离心4000转，2min，清洗两边。加入到前一天抗体孵育的细胞上清中继续4度摇2~4小时。这时候你可以制PAGE胶了。孵育结束后离心4000rpm，2min。弃去细胞上清，加入500ul TBS（1%土温-20）清洗2遍。再用无土温的TBS清洗一遍。（留下最后的清洗液，做Western。）加入上样Buffer，煮5分钟。上样，跑胶，转膜，封闭过夜。Day 6。进行剩余的Western步骤。（步骤略）注意事项：1。两个一抗一定要不同来源的，不然western的时候会有抗体杂带干扰。如果你的蛋白条带和抗体的重轻链差别很大，也可以不用。但这样就要有空抗体对照，比较浪费抗体。不建议采用。2。细胞裂解条件，一定要要4度进行，有冷库最好，没有的话，一定先预冷才能试验。加入高浓度的(protease inhibitor)，一定不能省。这个我是有教训的。3。内源性蛋白，细胞量一定要过量。4。不要省，一般优质2ug，普通抗体3~5ug时必须的。不要省坑体，否则会导致整个试验失败。不当之处，大家拍砖。斑竹，申请加2分哦
相关热词：
..........
生物秀是目前国内最具影响力的生物医药门户网站之一，致力于IT技术和BT的跨界融合以及生物医药领域前沿技术和成功商业模式的传播。为生物医药领域研究人员和企业提供最具价值的行业资讯、专业技术、学术交流平台、会议会展、电子商务和求职招聘等一站式服务。
官方微信号：shengwuxiu
电话：021-小木虫 --- 500万硕博科研人员喜爱的学术科研平台
&&查看话题
冷泉港分子克隆实验protocol
好东西就要分享
Table of Contents
Chapter 1: Plasmids and Their Usefulness in Molecular Cloning
Chapter 2: Bacteriophage and Its Vectors
Chapter 3: Working with Bacteriophage M13 Vectors
Chapter 4: Working with High-Capacity Vectors
Chapter 5: Gel Electrophoresis of DNA and Pulsed-Field Agarose
Chapter 6: Preparation and Analysis of Eukaryotic Genomic DNA
Chapter 7: Extraction, Purification, and Analysis of mRNA from Eukaryotic Cells
Chapter 8: In Vitro Amplification of DNA by the Polymerase Chain Reaction
Chapter 9: Preparation of Radiolabeled DNA and RNA Probes
Chapter 10: Working with Synthetic Oligonucleotide Probes
Chapter 11: Preparation of cDNA Libraries and Gene Identification
Chapter 12: DNA Sequencing
Chapter 13: Mutagenesis
Chapter 14: Screening Expression Libraries
Chapter 15: Expression of Cloned Genes in Escherichia coli
Chapter 16: Introducing Cloned Genes into Cultured Mammalian Cells
Chapter 1 Plasmids and Their Usefulness in Molecular Cloning
Protocol 1: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation
Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with alkali
Protocol 2: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation
Plasmid DNA is isolated from intermediate-scale (20-50 ml) bacterial cultures by treatment with
alkali and SDS.
Protocol 3: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation
Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with alkali
Protocol 4: Preparation of Plasmid DNA by Small-scale Boiling Lysis
Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with Triton X-
100 and lysozyme, followed by heating. This method is not recommended for preparing
plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains).
Protocol 5: Preparation of Plasmid DNA by Large-scale Boiling Lysis
Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with Triton X-
100 and lysozyme, followed by heating. This method is not recommended for preparing
plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains).
Protocol 6: Preparation of Plasmid DNA: Toothpick Minipreparation
Plasmid DNA is prepared directly from bacterial colonies plucked from the surface of agar
media with toothpicks.
Protocol 7: Preparation of Plasmid DNA by Lysis with SDS
Large (>15 kb), closed circular plasmids are prepared (albeit inefficiently and in small yield) by
lysing bacteria with SDS.
Protocol 8: Purification of Plasmid DNA by Precipitation with Polyethylene Glycol
Crude preparations of plasmid DNA are first treated with lithium chloride and RNase (to remove
RNA). The plasmid DNA is then precipitated in a solution containing polyethylene glycol and
Protocol 9: Purification of Plasmid DNA by Chromatography
The following table summarizes the salient features of many of the commercial resins that are
currently available for plasmid purification. Individual manufacturers supply detailed
instructions, which should be followed to the letter.
Protocol 10: Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-
Ethidium Bromide Gradients: Continuous Gradients
Solutions containing plasmid DNA are adjusted to a density of 1.55 g/ml with solid CsCl. The
intercalating dye, ethidium bromide, which binds differentially to closed circular and linear
DNAs, is then added to a concentration of 200 μg/ml. During centrifugation to equilibrium, the
closed circular DNA and linear DNAs form bands at different densities.
Protocol 11: Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-
Ethidium Bromide Gradients: Discontinuous Gradients
A solution containing plasmid DNA, saturating amounts of ethidium bromide, and CsCl (44%
w/v) is layered between two solutions of lesser (35% w/v CsCl) and greater density (59% w/v
CsCl). During centrifugation to equilibrium, the closed circular plasmid DNA and linear DNAs
form bands at different densities.
Protocol 12: Removal of Ethidium Bromide from DNA by Extraction with Organic
Ethidium bromide is removed from DNA by phase extraction with organic solvents.
Protocol 13: Removal of Ethidium Bromide from DNA by Ion-exchange Chromatography
Ethidium bromide is removed from DNA by chromatography through a cation-exchange resin.
Protocol 14: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid
DNA by Centrifugation through NaCl
Contamination of plasmid DNA by fragments of DNA and RNA is reduced to an acceptable
level by centrifugation through 1 M sodium chloride. This method was devised by Brian Seed
when he was a graduate student at Harvard University.
Protocol 15: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid
DNA by Chromatography through Sephacryl S-1000
Contamination of plasmid DNA by small fragments of nucleic acid is reduced dramatically by
size-exclusion chromatography through Sephacryl S-1000.
Protocol 16: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid
DNA by Precipitation with Lithium Chloride
High-molecular-weight RNA and proteins can be precipitated from preparations of plasmid
DNA by high concentrations of LiCl and removed by low-speed centrifugation.
Protocol 17: Directional Cloning into Plasmid Vectors
Directional cloning requires that the plasmid vector be cleaved with two restriction enzymes
that generate incompatible termini and that the fragment of DNA to be cloned carries termini
that are compatible with those of the doubly cleaved vector.
Protocol 18: Attaching Adaptors to Protruding Termini
Adaptors are short double-stranded synthetic oligonucleotides that carry an internal restriction
endonuclease recognition site and single-stranded tails at one or both ends. Adaptors are used
to exchange restriction sites at the termini of linear DNA molecules. They may be purchased in
phosphorylated and unphosphorylated forms.
Protocol 19: Blunt-ended Cloning into Plasmid Vectors
/members/chapter.jsp?chapter=112 (1 / 3)
Cold Spring Harbor Laboratory Press - Molecular Cloning - Chapter 1
Target DNA is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction
are used to transform competent E. coli. The maximum number of "correct" clones can
generally be obtained from ligation reactions containing equimolar amounts of plasmid and
target DNAs, with the total DNA concentration being <100 μg/ml. Blunt-end ligation catalyzed
by bacteriophage T4 DNA ligase is suppressed by high concentrations (5 mM) of ATP and
polyamines such as spermidine.
Protocol 20: Dephosphorylation of Plasmid DNA
During ligation in vitro, T4 DNA ligase will catalyze the formation of a phosphodiester bond
between adjacent nucleotides only if one nucleotide carries a 5&#180;-phosphate residue and the
other carries a 3&#180;-hydroxyl terminus. Recircularization of vector DNA can therefore be
minimized by removing the 5&#180;-phosphate residues from both termini of the linear, doublestranded
plasmid DNA with alkaline phosphatase.
Protocol 21: Addition of Synthetic Linkers to Blunt-ended DNA
Linkers are small self-complementary pieces of synthetic DNA, usually 8-16 nucleotides in
length, that anneal to form blunt-ended, double-stranded molecules containing a restriction site.
Linkers are used to equip blunt-ended termini of DNA with restriction sites as an aid to cloning.
Protocol 22: Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose
Ligation in low-melting-temperature agarose is much less efficient than ligation with purified
DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for
rapid subcloning of segments of DNA in dephosphorylated vectors and assembling
recombinant constructs.
Protocol 23: The Hanahan Method for Preparation and Transformation of Competent E.
coli: High-efficiency Transformation
This procedure generates competent cultures of E. coli that can be transformed at high
frequencies (5 x 108 transformed colonies/mg of superhelical plasmid DNA). IMPORTANT All
steps in this protocol should be carried out aseptically.
Protocol 24: The Inoue Method for Preparation and Transformation of Competent E.
Coli: "Ultra-Competent" Cells
This protocol reproducibly generates competent cultures of E. coli that yield 1 x 108 to 3 x 108
transformed colonies/mg of plasmid DNA. The protocol works optimally when the bacterial
culture is grown at 18°C. If a suitable incubator is not available, a standard bacterial shaker can
be set up in a 4°C cold room and regulated to 18°C.
Protocol 25: Preparation and Transformation of Competent E. coli Using Calcium
This protocol, developed approx. 30 years ago, is used to prepare batches of competent
bacteria that yield 5 x 106 to 2 x 107 transformed colonies/μg of supercoiled plasmid DNA.
Protocol 26: Transformation of E. coli by Electroporation
Electrocompetent bacteria are prepared by growing cultures to mid-log phase, washing the
bacteria extensively at low temperature, and then resuspending them in a solution of low ionic
strength containing glycerol. DNA is introduced during exposure of the bacteria to a short highvoltage
electrical discharge.
Protocol 27: Screening Bacterial Colonies Using X-gal and IPTG: -Complementation
-complementation occurs when two inactive fragments of E. coli -galactosidase associate
to form a functional enzyme. Many plasmid vectors carry a short segment of DNA containing
the coding information for the first 146 amino acids of -galactosidase. Vectors of this type are
used in host cells that express the carboxy-terminal portion of the enzyme. Although neither the
host nor the plasmid-encoded fragments of -galactosidase are themselves active, they can
associate to form an enzymatically active protein. Lac+ bacteria that result from -
complementation are easily recognized because they form blue colonies in the presence of the
chromogenic substrate X-gal. However, insertion of a fragment of foreign DNA into the
polycloning site of the plasmid almost invariably results in production of an amino-terminal
fragment that is no longer capable of -complementation. Bacteria carrying recombinant
plasmids therefore form white colonies. The development of this simple blue-white color test
has greatly simplified the identification of recombinants constructed in plasmid vectors.
Protocol 28: Screening Bacterial Colonies by Hybridization: Small Numbers
This procedure, a variant of the Grunstein and Hogness (1979) method, is used to screen a
small number of bacterial colonies (<200) that are dispersed over several agar plates and are
to be screened by hybridization to the same radiolabeled probe. The colonies are gridded onto
a master plate and onto a nitrocellulose or nylon filter laid on the surface of a second agar
plate. After a period of growth, the colonies on the filter are lysed and processed for
hybridization. The master plate is stored until the results of the screening procedure become
available.
Protocol 29: Screening Bacterial Colonies by Hybridization: Intermediate Numbers
Bacterial colonies growing on agar plates are transferred en masse to nitrocellulose filters. The
spatial arrangement of colonies on the plates is preserved on the filters. After transfer, the
filters are processed for hybridization to an appropriate radiolabeled probe while the original
(master) plate is incubated for a few hours to allow the bacterial colonies to regrow in their
original positions. This technique, a variant of the Grunstein and Hogness (1975) method, was
developed at Cold Spring Harbor Laboratory in 1975. The procedure works best with 90-mm
plates containing <2500 colonies.
Protocol 30: Screening Bacterial Colonies by Hybridization: Large Numbers
This procedure is used to plate, replicate, and subsequently screen large numbers of bacterial
colonies (up to 2 x 104 colonies per 150-mm plate or 104 colonies per 90-mm plate).
Protocol 31: Lysing Colonies and Binding of DNA to Filters
In this protocol, based on the procedure of Grunstein and Hogness (1975), alkali is used to
liberate DNA from bacterial colonies on nitrocellulose or nylon filters. The DNA is then fixed to
the filter by UV-cross-linking or baking under vacuum.
Protocol 32: Hybridization of Bacterial DNA on Filters
This protocol describes procedures to hybridize DNA from transformed colonies immobilized on
filters with radiolabeled probes and to recover from a master plate the corresponding colonies
that hybridize specifically to the probe.
Chapter 17: Analysis of Gene Expression in Cultured Mammalian Cells
Chapter 18: Protein Interaction Technologies
研究生必备与500万研究生在线互动！
扫描下载送金币微信公众平台测试账号,高级群发接口调用后提示成功,但微信用户并未收到消息
[问题点数：100分]
微信公众平台测试账号,高级群发接口调用后提示成功,但微信用户并未收到消息
[问题点数：100分]
不显示删除回复
显示所有回复
显示星级回复
显示得分回复
只显示楼主
2013年8月 .NET技术大版内专家分月排行榜第三2011年10月 .NET技术大版内专家分月排行榜第三
2013年8月 .NET技术大版内专家分月排行榜第三2011年10月 .NET技术大版内专家分月排行榜第三
2016年6月 .NET技术大版内专家分月排行榜第二2016年3月 .NET技术大版内专家分月排行榜第二2016年1月 .NET技术大版内专家分月排行榜第二2015年12月 .NET技术大版内专家分月排行榜第二2015年2月 .NET技术大版内专家分月排行榜第二2015年1月 .NET技术大版内专家分月排行榜第二2014年11月 .NET技术大版内专家分月排行榜第二2014年5月 .NET技术大版内专家分月排行榜第二2014年4月 .NET技术大版内专家分月排行榜第二2012年2月 多媒体/设计/Flash/Silverlight 开发大版内专家分月排行榜第二
2016年5月 .NET技术大版内专家分月排行榜第三2016年4月 .NET技术大版内专家分月排行榜第三2016年2月 .NET技术大版内专家分月排行榜第三2015年10月 .NET技术大版内专家分月排行榜第三2015年9月 .NET技术大版内专家分月排行榜第三2015年7月 .NET技术大版内专家分月排行榜第三2015年6月 .NET技术大版内专家分月排行榜第三2015年5月 .NET技术大版内专家分月排行榜第三2015年4月 .NET技术大版内专家分月排行榜第三2014年12月 .NET技术大版内专家分月排行榜第三2014年10月 .NET技术大版内专家分月排行榜第三2014年9月 .NET技术大版内专家分月排行榜第三2014年1月 .NET技术大版内专家分月排行榜第三2013年12月 .NET技术大版内专家分月排行榜第三2013年10月 .NET技术大版内专家分月排行榜第三2013年5月 .NET技术大版内专家分月排行榜第三2011年9月 .NET技术大版内专家分月排行榜第三2011年2月 .NET技术大版内专家分月排行榜第三2010年2月 .NET技术大版内专家分月排行榜第三
匿名用户不能发表回复！|
每天回帖即可获得10分可用分！小技巧：
你还可以输入10000个字符
(Ctrl+Enter)
请遵守CSDN，不得违反国家法律法规。
转载文章请注明出自“CSDN（www.csdn.net）”。如是商业用途请联系原作者。我做转染293，按着protocol，试了四次，转染效率只有10%，哪位高人指导一下啊(转染,培养基,培养液,质粒) - 细胞实验 - 生物秀
标题: 我做转染293，按着protocol，试了四次，转染效率只有10%，哪位高人指导一下啊(转染,培养基,培养液,质粒)
摘要: [我做转染293，按着protocol，试了四次，转染效率只有10%，哪位高人指导一下啊(转染,培养基,培养液,质粒)] protocol:1转染前24h，胰酶消化细胞，完全培养液1-5*105cell cm2,培养过夜，转染前两小时换液。2六块板300μl体系 A:2 5MCacl2取15μl,质粒1-5μg,无菌水补至150μl。
B:2*HepEs盐溶液150μL
把B放在vortex上，逐滴滴入A，滴完后对壁反复吹打100下左右3迅速弹管壁混匀，静置20min（出现细小颗 关键词:[转染 培养基 培养液 质粒 胰酶 荧光 培养皿]……
protocol:1转染前24h，胰酶消化细胞，完全培养液1-5*105cell/cm2,培养过夜，转染前两小时换液。2六块板300μl体系 A:2.5MCacl2取15μl,质粒1-5μg,无菌水补至150μl。
B:2*HepEs盐溶液150μL
把B放在vortex上，逐滴滴入A，滴完后对壁反复吹打100下左右3迅速弹管壁混匀，静置20min（出现细小颗粒）4 立即加入CELL培养皿中，混匀，立即加培养基，并发现培养基变黄（也可加入AB后，静置2min-15min再补加培养基）5培养12-16h后换液。（我在练转GFP）我六孔板加培养基500-800μl,算上300μl体系总体积800-1100μl。我培养24小时几乎看不到荧光，48h只能看到10%但亮度不高，的确小弟的293细胞状态不好，而且长到90%以上。
回复293细胞的转染，你为什么不用脂质体的转染呢，比如lipofectame2000等。钙磷转染法很考验操作者的经验，比如你的HEPES的pH值是否是最佳的，这个需要根据细胞实验进行调整的，还有你的HEPES是你自己配的吗，配了多久，以前有没有做过转染率的验证？钙磷混合时，需要观察混合液静置15分钟左右是否变浑浊然后再加入细胞内。半小时后观察细胞表面是否有一层细沙状的细小沉淀形成，把培养基弃掉，加入正常完全培养基。这样对细胞造成的毒性最小，同时保证了转染效率。
你的GFP质粒是你自己最近提取的吗，质粒的状态与转染率也有很大关系，GFP的量很少就可了，比如作为转染率指示时0.2ug就够了，一个6孔板的孔转染4 ug质粒。6孔板需要加入2ml培养基。
总之，如果经费允许的话，还是用脂质体的转染试剂吧，钙磷法太考验技巧了，有经验的人都不能保证每次都成功。回复我用3.5cm小皿练手，我加300μl体系，再补加500μl培养基，你觉得是不是太少了回复2*hepes是自己配的，我师兄用相同的一套东西，转染效率能达到30%，这样就可以做实验了回复我是用的脂质体做的转染。有一点我觉得要注意的是（质粒）的加入量，还有就是293细胞的状态。我在六孔板加的是8～10微克的。回复293的转染率到30%其实也是很低的，因为不到一半的转染率。这样的转染率可能会使后续实验得不到理想的结果。还是换成脂质体转染吧，除非有些细胞用脂质体很难转染，如原代的神经元细胞，也不要省这么一点钱了，时间成本会更高的。
相关热词：
..........
生物秀是目前国内最具影响力的生物医药门户网站之一，致力于IT技术和BT的跨界融合以及生物医药领域前沿技术和成功商业模式的传播。为生物医药领域研究人员和企业提供最具价值的行业资讯、专业技术、学术交流平台、会议会展、电子商务和求职招聘等一站式服务。
官方微信号：shengwuxiu
电话：021-}