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[交流]
做材料，不懂生物，请教分子生物学的同学
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这个实验室考察一些类二噁英对acetylcholinesterase (AChE）转录或者酶活性的影响的实验 Reporter Gene Construction, Transfection, and Luciferase Activity Determination pAChE-Luc and the mutated construct, pAChE-M-Luc (derived from pAChE-Luc), with mutated sequence on the 5′ putative DRE site were constructed as in Xie et al. (2013). The cells were seeded in 96-well plates at 10,000 cells/well 24 h before transient transfection using PolyJetTM reagent following the manufacturer's instructions (SignaGen Laboratories, Rockville, MD, USA). The Renilla luciferase encoding plasmid pRL-SV40 (Promega) was used as a normalization control, which was co-transfected with pAChE-Luc or pAChE-M-Luc into the cells. The mass ratio of target plasmid and control plasmid was 50:1. Twenty four hours thereafter, the cells were exposed to 1,2,3,7,8-PCDD, 2,3,7,8-TCDF, 2,3,4,7,8-PCDF, and 2,3,7,8-TBDD with toxic equivalent concentrations (TEQs) equivalent to 10−9 M of 2,3,7,8-TCDD. Twenty four hours after exposure, cells were washed with PBS and underwent luciferase activity determination, in which 100 μl of cell lysis buffer was added per well followed by a 10-min shake at room temperature for collecting the lysates. The lysates were then loaded in a spectrometer with automatic injection of luciferase substrate (TECAN Infinite F200 Pro). 我对上述实验过程和如何实验不了解，比如瞬时转染等，它是如何做到的 我对分子知道的很少，求解答啊 下面还有别的内容，供给我解答的人理解 Real-Time Quantitative PCR The total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was prepared using total RNA and Moloney Murine Leukemia Virus Reverse Transcriptase (Invitrogen). Real-time PCR was performed on equal amounts of cDNA using SYBR Green Master Mix and Rox reference dye (Promega). For human AChET transcript (the major AChE transcript in neurons, GenBank accession number NM_000665), the primers were 5′-GGG GTT CCC CAG GTA AGT GAC CT-3′ (forward) and 5′-TGA GCA GCG ATC CTG CTT GCT GTA G-3′ (reverse). For human 18S rRNA (NR_003286), the primers were 5′-GAA CGA GGA ATT CCC AGT AAG-3′ (forward) and 5′-GAT AGT CAA GTT CGA CCG TC-3′ (reverse). The SYBR and Rox signal was measured by the MX3005P quantitative PCR system (Stratagene, La Jolla, CA, USA). Amplification specificity was confirmed by 1 % agarose gel electrophoresis. ∆∆CT method was used to quantify the relative mRNA expression levels (Winer et al. 1999). Data Analysis SPSS software (version 16.0) was used to perform statistical tests. One-way analysis of variance (ANOVA) and multiple comparisons were used for the analysis of enzyme activity, promoter activity, and relative mRNA expression levels. p < 0.05 was considered to be statistically significant. |
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小木虫: 金币+0.5, 给个红包，谢谢回帖
markar: 金币+1, 赞一个！ 2017-01-10 20:06:46
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markar: 应助指数+1, 忘记加应助了！ 2017-01-10 20:07:24
小木虫: 金币+0.5, 给个红包，谢谢回帖
markar: 金币+1, 赞一个！ 2017-01-10 20:06:46
hopedream(markar代发): 金币+2, 替楼主感谢你！ 2017-01-10 20:07:02
markar: 应助指数+1, 忘记加应助了！ 2017-01-10 20:07:24
这个质粒是公司给构建好，转染就是买好转染试剂盒，按照说明书一混添加就行了。。。。看似过程简单，不同细胞转染效率却差别很大  发自小木虫Android客户端 |
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