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【共享】双酶切

1、 在双酶切载体时如果2个酶切位点靠得很近,必须注意酶切顺序。因为有的限制性内切酶要求其识别序列的两端至少保留有若干个碱基才能保证酶的有效切割。有的酶要求识别序列两端有多个碱基的,则必须先切,否则就可能造成酶切失败。2、 回收PCR产物:回收的PCR产物片段=1:10 ,一般取前者0.03pmol,后者取0.3pmol。pmol为单位的DNA转换为为?g单位的DNA:(X pmoles×长度bp×650)/ 1,000,000 (注:长度bp× ...

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【交流】关于PCR假阴性问题总结(建议大家看看)

关于PCR假阴性问题总结(建议大家看看)PCR的假阳性问题深受重视,但我个人认为PCR假阴性问题相对于临床检测更为严重。其实PCR假阳性的问题是比较单纯的,一般仅涉及污染和引物的非特异性问题。而污染可以通过严格的实验室管理,合理的环境设置,以及加入UNG酶抗污染基本可以解决,而引物的非特异性问题基本属于厂家试剂质量问题。而PCR的假阴性却不同,它涉及了与PCR实验的几乎所有人员和技术环节,十分复杂。就临床而言, ...

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【建议】RAW264.7细胞转染

不知道那些大侠做过,可以一起讨论一下.我试了几种转染试剂 ( JetpET, Lipofectine), 转染效率都极低 .只有电转的时候,转染效率也至多达到5%.不知道怎么拿这个细胞做luciferase.筛选浓度我已经用到1mg/ml了,不知道大家都用的多少?常规用FUGEGE6效果好点, 想效率高用逆转录病毒!"筛选浓度"你指什么? G418 我用过1200ug/ml, 1500ug/ml.对,就是指的G418.大侠你用过Fugene6吗?转染效率有 ...

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【共享】CopyControl克隆技术:一步法实现单拷贝到高拷贝

一般常识,单拷贝克隆产量低,高拷贝克隆稳定性差,一直让研究者在克隆表达时难以选择。蛋白的表达就有诱导表达系统,可以实现人为地控制蛋白的表达时间和表达量——现在连质粒的拷贝数可以借助诱导的方法来人为控制了!Epicentre的专利技术——CopyControl克隆系统能够让您共享单拷贝和高拷贝的优点。CopyControl克隆首先以单拷贝形式生长——单拷贝可以确保插入稳定及成功克隆编码毒性基因序列——在需要的时候, ...

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【资源】实验九 转化克隆的筛选和鉴定

1.目的学会用酶切法或PCR法筛选重组质粒转化成功的克隆菌。2.原理利用转化后宿主菌所获得的抗菌素抗性性状筛选出获得质粒的菌落克隆。再从这些菌落中鉴定出质粒上带有外源基因插入片段的克隆菌落。3.器材旋涡混合器,小镊子,微量移液取样器,移液器吸头,1.5ml 微量离心管,双面微量离心管架,干式恒温气浴(或恒温水浴锅),制冰机,恒温摇床,超净工作台,酒精灯,无菌牙签,摇菌管。4.试剂LB培养基(加抗菌素),PCR用试剂,引物,质粒提 ...

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请教PCR

我想请教大家:我的PCR跑出四条带,亮度不相上下,包括目的基因,其余杂带比目的带短,退火温度52-58都试过,没改善。接着调高退火温度有可能得到特异条带吗?会不会引物设计有问题?我用的是PRIMER DESIGNER 1.01.烦劳看看我的基因序列,引物,要真有问题,就死心塌地重订引物了。1 cggcagcagc gggaggcgag gggcgccacc gaggagccga gcccgccgag ccgggcgctc61 tatttcagcg gg ...

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【共享】酵母电转改进PROTOCOL

小弟弟一次做电转,完全按照毕氏酵母说明书来,居然没长出来。第二次,按老板出国前留下的做,效果甚好!现贡献出来,望对大家有所帮助!小弟刚做酵母表达,在DXY受益匪浅!看到不少关于酵母电转的PROTOCOL,但基本上是按INVITROGEN上的步骤来的。小弟第一次也是照本宣科,可是没出来,后来又按导师留给小弟的PROTOCOL做了一次,转化效果特好。先将其贡献给大家,希望对大家有所帮助:Yeast Transformati ...

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【共享】简明扼要的一些重要分子生物学概念(in English)!

Nucleic AcidsGel electrophoresisAgarose Gel ElectrophoresisPulse-field Gel ElectrophoresisPolyacrylamide Gel ElectrophoresisRestriction Enzyme (RE) DigestionWhat are RE?Enzymes that cut dsDNA, usually palindromic sequences. RE recognize 4-8bp.A RE that ...

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【共享】Forensic DNA Typing, Second Edition: Biology, Technology, and Genetics of STR Markers电子书下载

Forensic DNA Typing, Second Edition: Biology, Technology, and Genetics of STR MarkersBook Description:Since the enormously successful first edition of Forensic DNA Typing was published, the Human Genome Project has published a draft sequence of the human genome and completed the fin ...

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【bio-news】通过二代测序对癌症基因组认识的进展

Advances in understanding cancer genomes through second-generation sequencingCancers are caused by the accumulation of genomic alterations. Therefore, analyses of cancer genome sequences and structures provide insights for understanding cancer biology, diagnosis a ...

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【专题讨论】外文文献讨论(一)---------欢迎大家积极参与、共同提高

本人的想法:拿出自己研究过的文献(外文),供大家提出问题,讨论、翻译。相信大家的讨论一定会碰撞出新的ideas来。(已经搜索了相关内容,应该不是重复法帖,请班主支持!)相互交流、享受提高!!!Small Interfering RNA-Mediated Gene Silencing in TLymphocytes1Michael T. McManus, Brian B. Haines, Christopher P. Dillon, Charles E. Whitehurst,Luk van Par ...

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some words about how to improve your research ability

When I read some posts, I often read some very basic questions. I always thinking, these questions are commoly taught by attending master or Ph.D courses. Or is easily solved by their tutors. Why they still ask such questions? So I write some of my thinkings, it may be benificial to new beginers. Due to my limited ti ...

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赶超RNAi的基因沉默技术? [zt from dnathink]

The Scientist-September 23,2003-Fingering single genes-Cathy HoldingThe ability to control in vivo gene expression may be a powerful tool in cancer therapy, gene therapy of inherited genetic diseases, and for the study of gene function. Small interfering RNAs are increasingly used in ...

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【资源】Methods In Molecular Biology Collection - Volumes 101-200

缺9卷Methods In Molecular Biology Collection - Volumes 101-200101-Mycobacteria Protocols-Methods in Mol Biol Vol 101.pdf102-Bioluminescence Methods and Protocols-Methods in Mol Biol Vol 102.pdf103-Pichia Protocols-Methods in Mol Biol Vol 103.pdf104-Mycoplasma Pro ...

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[小常识]Restriction Endonucleases: An Overview

Restriction Endonucleases: An OverviewRestriction enzymes were discovered about 30 years ago during investigations into the phenomenon of host-specific restriction and modification of bacterial viruses. Bacteria initially resist infections by new viruses, and this "re ...

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【资源】大肠杆菌的表型大全

刚刚看到的,基本上各种常见的和不常见的大肠杆菌表型在下面都可以查到,内容不是我写的,想成为分子生物学高手的网友应该好好研究一下。其内容来自:http://openwetware.org/wiki/E._coli_genotypesF- = Does not carry the F plasmidF+ = Carries the F plasmid. The cell is able to mate with F- through conjugation.F' = Carries an F plasmid that has host chr ...

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Genome Informatics

http://www.jsbi.org/journal.htmlAt this first year of the new millennium, GIW has changed its name to the ``International Conference on Genome Informatics,'' while keeping GIW as its acronym. In the ``post-genome'' era, after the completion of the draft sequence of the human genome, we are faced ...

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【共享】2014年SCI影响因子新鲜出炉!

北京时间2014年7月30日,汤森路透《SCI期刊分析报告》(Journal Citation Reports)新鲜出炉,该份报告涵盖82个国家237个大类10927本期刊。今年公布的是这些杂志2013年的影响因子,今年新增了379种期刊,同时也剔除了33种期刊,因为它们自引率过高。影响因子在一定程度上的确是一本杂志质量高低的标准之一,并且能够带来科学以外太多的东西:教职、基金申请、学术影响力等。尽管很多学者批评过杂志的 ...

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PET技术在基因治疗应用中的一篇文献

Mol Imaging. 2005 Oct-Dec;4(4):463-72.Micro-PET/CT monitoring of herpes thymidine kinase suicide gene therapy in a prostate cancer xenograft: the advantage of a cell-specific transcriptional targeting approach.Johnson M, Sato M, Burton J, Gambhir SS, Carey M, Wu L.University of Cali ...

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【共享】关于核酸和蛋白的一些换算

转自:http://www.genscript.com/conversion.html一,Spectrophotometric Conversions1 A260 unit of double-stranded DNA=50 µg/ml1 A260 unit of single-stranded DNA=33 µg/ml1 A260 unit of single-stranded RNA=40 µg/ml二,DNA Molar Conversions1 µg of 1,000 bp DNA =1.52pmol(3.03pmol ...

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