Molecular and Cellular Biology Techniques
分子和细胞生物学技术
基本信息
- 批准号:7140015
- 负责人:
- 金额:$ 18.89万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2006
- 资助国家:美国
- 起止时间:2006-01-01 至 2010-12-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
Core D provides technical expertise and state of the art molecular and cellular biology methodology for
cloning and expression of the proteins described in the program project. This centralized operation coordinates
all molecular biology work and ensures that all proteins are cloned and expressed in a uniform
fashion. This is particularly important since several investigators plan to analyze different aspects of some of
the same proteins. This uniformity greatly enhances accuracy of comparison between the proteins.
Core D designs and clones expression vectors encoding wild type or mutated proteins as requested by the
individual investigators. All wild type genes described in this program project are identified, sequenced and
assembled in cloning vectors. Thus, vectors expressing wild type or mutated proteins can be cloned from
cDNAs. Vectors expressing consensus sequence peptides will be constructed from synthetic
oligonucleotides. Depending on the requirement for the analysis of the protein (amount and/or modification of
protein), the expression vectors are designed for expression in prokaryotic or eukaryotic cell lines. Core D
also establishes cell lines expressing the proteins, determines conditions for optimum protein expression and
aids in the purification of the expressed protein.
Thus the service of Core D includes: a) advice toward an optimal expression system, b) design
methodologies to express desired proteins, c) confirm wild type DNA sequence by restriction and/or DNA
sequence analysis, d) design primers for DNA sequence analysis, e) design primers for PCR amplification of
wild type or mutant constructs, f) extract DNA, g) digest DNA with restriction enzymes, h) amplify DNA by
PCR, i) ligate DNA (fragments) in cloning and expression vectors, j) transform/transfect ligated DNA into cell
lines to express the proteins, k) select clones, I) amplify clones, m) isolate permanently transfected cell lines,
n) maintain transformed cell lines, o) establish optimum protein expression conditions, p) assist in protein
purification, q) maintain cell lines carrying wild type and expression vectors.
All personnel in Core D trains new users associated with the projects in molecular biology and cell culture
techniques and oversees proper use of cell culture facilities. This ensures safe maintenance of cell systems
and guarantees compliance with NIH guidelines.
Core D designs and clones expression vectors encoding proteins requested by the principal investigators of
the individual projects. They establish cell lines, determine optimum conditions for protein expression and aid
in the purification of the expressed protein.
核心D提供技术专长和最先进的分子和细胞生物学方法,
克隆和表达的蛋白质中所描述的程序项目。这种集中的运作协调
所有的分子生物学工作,并确保所有的蛋白质克隆和表达在一个统一的
时尚.这一点特别重要,因为几位研究人员计划分析一些
相同的蛋白质。这种均匀性大大提高了蛋白质之间比较的准确性。
核心D设计和克隆编码野生型或突变蛋白的表达载体,如本发明所要求的。
个别调查员。本项目中描述的所有野生型基因都经过鉴定、测序,
在克隆载体中组装。因此,表达野生型或突变蛋白的载体可以从
cDNA。表达共有序列肽的载体将由合成的多肽构建。
寡核苷酸根据蛋白质分析的要求(蛋白质的量和/或修饰),
蛋白),表达载体被设计用于在原核或真核细胞系中表达。内核D
还建立表达蛋白质的细胞系,确定最佳蛋白质表达的条件,
有助于表达蛋白的纯化。
因此,核心D的服务包括:a)对最佳表达系统的建议,B)设计
c)通过限制性内切酶和/或DNA测序确认野生型DNA序列,
序列分析,d)设计用于DNA序列分析的引物,e)设计用于PCR扩增
野生型或突变体构建体,f)提取DNA,g)用限制酶消化DNA,h)通过
PCR,i)将DNA(片段)连接到克隆和表达载体中,j)将连接的DNA转化/转染到细胞中
表达蛋白质的细胞系,k)选择克隆,I)扩增克隆,m)分离永久转染的细胞系,
n)维持转化的细胞系,o)建立最佳的蛋白质表达条件,p)辅助蛋白质表达,
纯化,q)维持携带野生型和表达载体的细胞系。
核心D的所有人员培训与分子生物学和细胞培养项目相关的新用户
技术和监督细胞培养设施的正确使用。这确保了细胞系统的安全维护
并保证遵守NIH指南。
核心D设计和克隆编码蛋白质的表达载体,
个别项目。他们建立细胞系,确定蛋白质表达的最佳条件,
表达蛋白的纯化。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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MARGARETHA CARRAWAY其他文献
MARGARETHA CARRAWAY的其他文献
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{{ truncateString('MARGARETHA CARRAWAY', 18)}}的其他基金
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