Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine. In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host ...
Wang, J, Xu, R & Liu, A. (2014) IRDL cloning: a one-tube, zero-background, easy-to-use, directional cloning method improves throughput in recombinant DNA preparation. PLoS ONE. 2014 Sep 23; 9(9):e107907. PM ID: ...
The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction ...
For positive-selection cloning of PCR products through blunt-end ligation. Allows direct cloning of PCR products without purification.. GenHunters PCR-TRAP® Cloning System is the most efficient method for cloning PCR products by blunt-end ligation. This system uses a third generation cloning vector that features positive selection for cloning PCR products (see figures next page). Only recombinant plasmids confer antibiotic resistance, making PCR-TRAP® extremely efficient. There is no need for any post-PCR manipulation before cloning, since a significant fraction of PCR products do not contain the 3 overhanging A (Clark, 1988, Nucleic Acids Res. 16:9677 ...
TY - JOUR. T1 - Molecular cloning and primary structure of Man9‐mannosidase from human kidney. AU - BAUSE, Ernst. AU - BIEBERICH, Erhard. AU - ROLFS, Andreas. AU - VÖLKER, Christof. AU - SCHMIDT, Bernhard. PY - 1993/10. Y1 - 1993/10. N2 - Man9‐mannosidase, a processing enzyme found in the endoplasmic reticulum (ER), catalyses the removal of three distinct mannose residues from peptide‐bound Man9‐GlcNAc2 oligosaccharides producing a single Man6 isomer [Bause, E., Breuer, W., Schweden, J., Roesser, R. & Geyer, R. (1992) Eur. J. Biochem. 208, 451-457]. We have isolated four Man9‐mannosidase‐specific clones from a human kidney cDNA library and used these to construct a full‐length cDNA of 3250 base pairs. A single open reading frame of 1875 nucleotides encodes a protein of approximately 71 kDa, consistent with data from immunological studies. Analysis of the coding sequence predicts that Man9‐mannosidase is a type II transmembrane protein consisting of a short cytoplasmic ...
View Notes - PCR from BME 3406 at University of Florida. Cloning genes Cloning results in the purification of a single fragment of DNA from exceedingly complex DNA molecules. Need DNA corresponding
PCR Cloning Protocols, moment version, updates and expands Bruce Whites best-selling PCR Cloning Protocols (1997) with the latest methods for DNA cloning and mutagenesis. right here the researcher will locate with no trouble reproducible equipment for all of the significant points of PCR use, together with PCR optimization, desktop courses for PCR primer layout and research, and novel adaptations for cloning genes of distinct features or starting place, with emphasis on lengthy distance PCR and GC-rich template amplification. additionally integrated are either traditional and novel enzyme-free and restrict site-free methods to clone PCR items right into a diversity of vectors, in addition to state of the art protocols to facilitate DNA mutagenesis and recombination, and to clone the difficult uncharacterized DNA flanking a recognized DNA fragment. ...
Nucleic acids can be isolated from cells for the purposes of further analysis by breaking open the cells and enzymatically destroying all other major macromolecules. Fragmented or whole chromosomes can be separated on the basis of size by gel electrophoresis. Short stretches of DNA or RNA can be amplified by PCR. Southern and northern blotting can be used to detect the presence of specific short sequences in a DNA or RNA sample. The term cloning may refer to cloning small DNA fragments (molecular cloning), cloning cell populations (cellular cloning), or cloning entire organisms (reproductive cloning). Genetic testing is performed to identify disease-causing genes, and gene therapy is used to cure an inheritable disease.. Transgenic organisms possess DNA from a different species, usually generated by molecular cloning techniques. Vaccines, antibiotics, and hormones are examples of products obtained by recombinant DNA technology. Transgenic plants are usually created to improve characteristics of ...
The pUC18 plasmid and pUC19 plasmid enable successful cloning of larger DNA fragments than the M13 mp18 RF Phage Vector. Because these cloning vectors contain a multiple cloning site at the lacZ region, recombinant plamids can be verified via blue/white colony screening using agar plates containing IPTG and X-Gal. Expression of target DNA is enabled by the presence of a lac promoter in the cloning vectors.. ...
Use SimVector to draw plasmid maps, perform restriction analysis and mapping. The plasmid drawing software also simulates cloning experiments such as gateway cloning, ta cloning and restriction cloning
TY - JOUR. T1 - Molecular cloning and functional expression of rat leukotriene A4hydrolase using the polymerase chain reaction. AU - Makitala, Naomasa. AU - Funku, Colin D.. AU - Imaic, Enyu. AU - Hoover, Richard L.. AU - Badra, Kamal F.. PY - 1992. Y1 - 1992. N2 - We isolated a cDNA encoding rat leukotriene A4 (LTA4) hydrolase from mesangial cells by the polymerase chain reaction according to the human amino acid sequence. The deduced amino acid sequence shows that rat LTA, hydrolase is a 609 amino acid protein with an M, 69 kDa. Comparison of human LTA4 hydrolase revealed 93% homology, and include zinc-binding motifs of aminopeptidases. COS-7 cells transfected with the cDNA revealed substantial LTA4 hydrolase activity, and their activities were abolished by preincubation with captopril, representing the first reported cDNA expression of recombinant enzyme in mammalian cells. RNA blot analysis indicated that LTA4 hydrolase was expressed in glomerular endothelial, epithelial and mesangial ...
A bacterial cloning system for mapping and analysis of complex genomes has been developed. The BAC system (for bacterial artificial chromosome) is based on Escherichia coli and its single-copy plasmid F factor. It is capable of maintaining human genomic DNA fragments of greater than 300 kilobase pairs. Individual clones of human DNA appear to be maintained with a high degree of structural stability in the host, even after 100 generations of serial growth. Because of high cloning efficiency, easy manipulation of the cloned DNA, and stable maintenance of inserted DNA, the BAC system may facilitate construction of DNA libraries of complex genomes with fuller representation and subsequent rapid analysis of complex genomic structure.. ...
pEASY®-Blunt Zero Cloning Kit,Cloning Vectors,Cloning and Mutagenesis System,Products,Beijing TransGen Biotech Co.Ltd,OverviewContents& storageCitations & referencesRelated ImagesDownloadOverviewDescriptionpEASY ®-Blunt Zero Cloning Vector c
Puzzling Cloning Problem With a Specific Vector - posted in Molecular Cloning: Hello, over the last couple of months I have been attempting to generate artificial aggrobacterial vectors containing DNA inserts that correspond to the tripartite genome of the Cowpea Chlorotic Mosaic Virus. These vectors will be used to generate mutant virions in-Plantae. I am using a vector provided to our lab by a collborator. I must use this vector for all cloning experiments. Its a large vector ( abou...
CopyControl cDNA, Gene and PCR Cloning Kit w/ Chemically Competent E. coli cells from EPICENTRE Biotechnologies,The CopyControl PCR Cloning Kits are designed to speed up the PCR cloning process and to ensure that all PCR products, regardless of sequence or type of polymerase used, are efficiently cloned. All PCR products including those that are difficult to clone by other PCR cloning methods or that m,biological,biology supply,biology supplies,biology product
Cloning a foreign gene into E-coli - posted in Molecular Cloning: Dear Friends, I am facing the problem with cloning. I have insert with 2.6Kb to be cloned into 13Kb E-coli vector . Vector has cloned strong promoter which is derived from the source same as the insert coming from. According to the strategy the desired will be cloned in front of promoter using PCR product digested either with same enzyme at both the ends(NcoI) or two enzymes(NcoI and SmaI). After digestion,ligation and tra...
For every Herceptin, for every Enbrel, Humira and Rituxan, and for every successful biotherapeutic product in the market today, there is a long history of its product development. We all hope that our target protein product will make a difference in both the therapeutic market and the lives it will eventually affect as it reaches its ultimate goal of becoming a human therapeutic. The reality is, in order for it to be a feasible therapeutic, the protein must first be manufactured. And one of the very first steps in determining the manufacturing capability of the product is the crucial step of choosing the production cell line that will produce this protein.. There are many challenges in single cell CHO cloning procedures to obtain valuable stable CHO production cell lines. We often rely on efficient cloning strategies and cloning media to execute such a crucial task in the developmental process of a recombinant protein product. An important aspect of cloning strategies is the cloning media ...
1. A degenerate polymerase chain reaction (PCR) homology screening procedure was applied to rat brain cDNA in order to identify novel genes belonging to the amiloride-sensitive Na+ channel and degenerin (NaC/DEG) family of ion channels. A single gene was identified that encodes a protein related to …
The biosynthesis of steroid hormones is an integral component of insect growth, development and reproduction. Although there is an abundance of biochemical data implicating both microsomal and mitochondrial cytochrome P450s in steroid metabolism, molecular genetic information on mitochondrial P450s is almost entirely limited to vertebrate sequences. In the current study, a degenerate polymerase chain reaction (PCR) primer was targeted to the highly conserved region of P450 genes that encodes the heme-binding decapeptide. Using a 5 rapid amplification of cDNA ends (RACE) approach, seven novel cytochrome P450 genes were isolated from Drosophila acanthoptera, including one sequence (CYP12B1) with high regional homology to vertebrate mitochondrial P450s. Sequence analysis of the conceptual translation of the full length gene, obtained by 5RACE, revealed an amphipathic NH2-terminus rich in basic and hydrophilic amino acids, a characteristic feature of mitochondrial P450s that distinguishes them ...
PRODUCTS | Cloning & Gene Analysis | KOD -Plus- Mutagenesis Kit | The category leader, continuing to create new value that contributes to society in the environment, healthcare, and high-function product fields.
During the last few decades, techniques for manipulating eukaryotic as well as prokaryotic DNA have witnessed a remarkable development. There are three main phases of the development of these techniques, which include recombinant DNA technology and gene cloning; polymerase chain reaction and DNA chips and microarrays. Using recombinant DNA technology, we can isolate and clone single copy of a gene or a DNA segment into an indefinite number of copies, all identical. This became possible, because bacteria, phages and plasmids reproduce in their usual style, even after insertion of foreign DNA, so that the inserted DNA also replicates faithfully with the parent DNA. This technique is called gene cloning. It involves the production of a large number of identical DNA molecules from a single ancestral DNA molecule. The essential characteristic of gene cloning is that the desired gene or DNA fragments must be selectively amplified resulting in a large increase in copy number of selected DNA sequences. ...
Gentaur molecular products has all kinds of products like :search , GenTarget \ pEco-T7-nHis PCR cloning kit: PCR cloning kit with a built-in vector (T7 promoter based) in provided cloning cells for E Coli expression of N-term His-tagged protein. \ IC-1001 for more molecular products just contact us
Semi-solid cloning of mammalian cells in 96-well plates using ClonaCell™ FLEX is an efficient cloning method that enables selective expansion of only those clones producing the protein of interest. Whether cloning CHO cells, hybridomas or other cell
Reverse genetics is used in many laboratories around the world and enables the creation of tailor-made influenza viruses with a desired genotype or phenotype. However, the process is not flawless, and difficulties remain during cloning of influenza gene segments into reverse genetics vectors (pHW2000, pHH21, pCAGGS). Reverse genetics begins with making cDNA copies of influenza gene segments and cloning them into bi-directional (pHW2000) or uni-directional plasmids (pHH21, pCAGGS) followed by transfection of the recombinant plasmid(s) to HEK-293 T or any other suitable cells which are permissive to transfection. However, the presence of internal restriction sites in the gene segments of many field isolates of avian influenza viruses makes the cloning process difficult, if employing conventional methods. Further, the genetic instability of influenza gene-containing plasmids in bacteria (especially Polymerase Basic 2 and Polymerase Basic 1 genes; PB2 and PB1) also leads to erroneous incorporation of
Molecular cloning and deduced amino acid sequences of the alpha- and beta- subunits of mammalian NAD(+)-isocitrate dehydrogenase.: A 153 bp fragment of the cDNA
The procedure of PCR cloning includes DNA preparation, digestion, ligation and transformation. This video shows the first part- insert PCR. Compared to other Taq-generated PCR inserts, Pfu DNA polymerase-generated PCR fragments will have fewer errors and is more commonly used for molecular cloning. - PCR Cloning (I) - Inserted Fragment Preparation - AbVideo™ - Support - Abnova
P-57. Generating and Sequencing Full-length cDNAs of Novel Human Genes Within the German cDNA Consortium. Ruth E. Wellenreuther, Stefan Wiemann, Daniela Heiss, Nina Claudino, Annemarie Poustka, Department of Molecular Genome Analysis, German Cancer Research Center, Im Neuenheimer Heidelberg, GERMANY. We generate human cDNA libraries that are enriched in full length clones i.e. from the translation start to the poly A tail. These libraries are used for a) systematic sequencing within the cDNA consortium of the Genome Project aiming at the identification and analysis of as many new genes as possible and b) for screening to isolate full length clones of partial genes. Libraries are created by directional cloning of cDNAs into plasmid vectors. Full-length enrichment is achieved via Clontechs SMART technology. This method is PCR-based, and in our modified strategy, we amplify and clone selective size windows of the cDNA fraction above 3 kb.Clones from the libraries generated within this project are ...
We evaluated the cloning efficiency of different size PCR products into three T-vector cloning systems:. the pGEM-T and pGEM-T Easy Vector Systems and the TOPO TA Cloning System.
Learn about Molecular Cloning. Topics cover isolation of gene fragment, screening, polymerase chain reaction, cloning vectors, and the isolation of plasmids.
Get fast and efficient molecular cloning with SBI-quickly and easily make a range of constructs or let SBI take care of making them for you
The complete nucleotide and amino acid sequence of GP-2 was determined initially by molecular cloning technique from peptide sequence for rat, dog and humans (12, 13, 18, 43). The cDNA encodes a protein of 530 aa in humans with 67% identity to rat and 72% to dog GP-2. The primary sequence encodes 10 (human) or 8 (dog) Asn-linked glycosylation sites. This is consistent with data for in vitro translation by reticulocyte lysate that yields a protein of 55 kDa and treatment with N-glycanase which reduces the molecular mass of canine GP-2 from 75 kDa to 52 kDa (12, 18). Rat GP-2 has been shown to possess 5 or 6 N-linked high mannose carbohydrate chains (16). The primary protein sequence is also post-translationally glycosylated on its carboxyl terminal to a GPI moiety in the membrane during passage through the Golgi to reach the ZG (11, 13, 17). This GPI moiety contains a phosphoethanolamine linker attached to the protein, a 4-5 sugar core and a phosphatidylinositol whose hydrocarbon chains insert ...
Andre SzyszkowskiPsy 13004/28/05 Cloning: Choice is Ethical Thousands of people a year are placed on the organ donors list. Thousands of people a year are diagnosed with diseases that are dubbed fatal unless a transplant or transfusion is given. This has created a large demand for some alternative method to the present donor practice. Research in the taboos cience of cloning seems to provide a viable method in which to aid the problem aforementioned and many others as well. But is it ethical? Cloning technology is expected to aid the result in several medical breakthroughs. It is thought that there may one day be a cure for cancer. This is because the cloning process helps us understand the process of cell differentiation. Theories exist that if a cure for cancer can be found, then further testing may lead to a cure for heart attacks and cloning organs for organ transplantation. Scientists believe that they may be able to treat heart attack victims by cloning their healthy heart cells and ...
44.38935 -68.20742 BAR HARBOR, Maine - New scientific evidence has heightened concerns about the safety of cloning human beings, an international authority warned yesterday.. We now believe that the potential to make a normal human baby with cloning is almost zero, said Dr. Rudolf Jaenisch, professor of biology at the Massachusetts Institute of Technology, at a genetics conference at Maines Jackson Laboratory.. Cloning a normal human is an illusion. There are major problems, and the problems are very serious.. Dr. Jaenisch, who helped pioneer gene transfer and cloning, said the public continues to be enthralled by stories of maverick doctors who supposedly plan to clone human beings.. Italian doctors in Rome, for instance, claim that they will clone a baby by next spring, with $500,000 provided by a couple whose infant died after heart surgery. An American firm called Clonaid has made repeated claims about a imminent human cloning.. Cloning a normal baby is utter nonsense, said Dr. ...
Introduction. Does cloning benefit or endanger society? Marley Gibbons-Balfour Case Study: Biology Contents Introduction 1 Background Science 2 Arguments against 4 Arguments for 7 Summary 10 Conclusion 11 Bibliography 12 Introduction Cloning has quickly become one of the most contentious issues in modern society, along with other issues like abortion, homosexuality and euthanasia. Due to the conflicted teachings and ideologies of many people in the world, there is no general consensus about cloning. Some people feel that it could benefit humans (through cures, through solving infertility and through knowledge), while others feel it could endanger humans and is a bad thing (due to ethical issues and due to being unaware of what could happen if it didnt work). Because of this, I have decided to investigate whether cloning actually does benefit or endanger society. I will go about this by collecting 6 sources (3 against cloning and 3 for cloning) and evaluating the evidence they present with the ...
The cloning of a novel murine gene that encodes a protein with lymphopoietic activity is described. The activity was initially identified in a coculture system of an adherent thymic stromal cell line with a medullary phenotype, Z210R.1, and day 15 fetal liver (6). Interestingly, the nonadherent lymphoid-like cells that grew in these cocultures phenotypically resembled B cells. A clonal lymphoid line (NAG8/7) was derived from long term cocultures, and this line expresses B220, 6C3 (BP-1), and is surface μ+, but κ chain negative. Importantly, NAG8/7 cells proliferated in response to conditioned medium from the Z210R.1 cell line and thus became the basis of a bioassay that allowed for the preliminary characterization of the growth factor and the eventual cloning of the cDNA encoding this activity.. The cDNA encoding the NAG8/7 growth activity was cloned from a library made from the Z210R.1 cell line. Based on the nucleotide sequence, the mature TSLP protein consists of 121 amino acids with 3 ...
Gateway compatible bacterial expression vector with T7 promoter, N- and C-terminal His tags and thrombin cleavage site; contains ccdB death cassette that is removed after recombinational cloning; kanamycin resistance in bacteria; recombinational cloning ...
The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full‐length cDNAs in species‐specific expression vectors and subsequent functional analysis of the expressed protein
In-Fusion EcoDry PCR Cloning Kits allow you to clone any PCR fragment into any linearized vector in a single step without restriction digestion of the PCR fragment, ligation or blunt-end polishing.
A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during procedures involving molecular cloning or subcloning. Extremely useful in biotechnology, bioengineering, and molecular genetics, MCSs let a molecular biologist insert a piece of DNA or several pieces of DNA into the region of the MCS. This can be used to create transgenic organisms, also known as genetically modified organisms (GMOs). One bacterial plasmid used in genetic engineering as a plasmid cloning vector is pUC18. Its polylinker region is composed of several restriction enzyme recognition sites, that have been engineered into a single cluster (the polylinker). It has restriction sites for various restriction enzymes, including EcoRI, BamHI and PstI. Another vector used in genetic ...
TY - JOUR. T1 - Construction of a set of Saccharomyces cerevisiae vectors designed for recombinational cloning. T2 - Author correction. AU - Van Mullem, Vincent. AU - Wery, Maxime. AU - De Bolle, Xavier. AU - Vandenhaute, Jean. PY - 2004/1/30. Y1 - 2004/1/30. UR - http://www.scopus.com/inward/record.url?scp=1242273822&partnerID=8YFLogxK. U2 - 10.1002/yea.1064. DO - 10.1002/yea.1064. M3 - Comment/debate. AN - SCOPUS:1242273822. VL - 21. JO - Yeast. JF - Yeast. SN - 0749-503X. IS - 2. ER - ...
Both the colony formation rate (number of colonies) and the ratio of correct clones (cloning efficiency) in transformation are important determinants for efficient cloning of PCR fragments. Cell lysates from E. coli RecA− strains such as DH10B contain endogenous in vitro homologous recombination activity, and can be used to clone PCR fragments into vectors with homology regions. However, cloning with lysates from this strain is not efficient, particularly in the case of inserts with short homology lengths (approximately 15-20 bp), because of a lower colony formation rate [14]. An E. coli PPY strain that expresses an optimized λ prophage Red/ET recombination system circumvents this problem by increasing the colony formation rate during PCR fragment cloning [14]. To extend the utility of this method, I prepared SLiCE extracts from several E. coli laboratory strains with some modifications, and estimated the efficiency with which redox-related genes from Arabidopsis could then be cloned into ...
A method is provided for isolating and identifying a recombinant clone having a DNA segment therein coding for at least one desired heterologous polypeptide, at least a short amino acid sequence of which is known, by effecting cDNA synthesis on a mixture of mRNAs containing the mRNA coding for the desired polypeptide, isolating the resultant cDNA mixture, inserting the resultant cDNA into recombinant cloning vehicles, transforming hosts with the vehicles, separating the transformants and isolating and identifying a recombinant clone containing a DNA segment which is homologous over at least a portion thereof to at least one oligonucleotide probe specific for the DNA segment; wherein the probe is an extension of the nucleotide sequence of an oligonucleotide primer having a nucleotide sequence complementary to a region of the target mRNA coding for a portion of the known amino acid sequence, and is complementary to a longer region of the target mRNA coding for a longer portion of the known amino acid
A method is provided for isolating and identifying a recombinant clone having a DNA segment therein coding for at least one desired heterologous polypeptide, at least a short amino acid sequence of which is known, by effecting cDNA synthesis on a mixture of mRNAs containing the mRNA coding for the desired polypeptide, isolating the resultant cDNA mixture, inserting the resultant cDNA into recombinant cloning vehicles, transforming hosts with the vehicles, separating the transformants and isolating and identifying a recombinant clone containing a DNA segment which is homologous over at least a portion thereof to at least one oligonucleotide probe specific for the DNA segment; wherein the probe is an extension of the nucleotide sequence of an oligonucleotide primer having a nucleotide sequence complementary to a region of the target mRNA coding for a portion of the known amino acid sequence, and is complementary to a longer region of the target mRNA coding for a longer portion of the known amino acid
There have been several successes of cloning especially the birth of Dolly the sheep in 1997, which lived for six years. Nuclear transfer involves the fusion of somatic cells and enucleated egg cells. Cloning of pig cells will be the focus of this paper. Scientists have identified pig clones as a potential hope for the future because of the possibility of xenotransplantation. Xenotransplantation has resulted from the merging of cloning with an additional biotechnology technique of genetic engineering. In addition, cloning has led to new prospects of livestock breeding and advances in medical procedures. This paper will discuss the procedure involved in the cloning of pig cells. Cloning is a multi-step process that scientists have endeavored to advance for a long time. The success story of Dolly the sheep served as a breakthrough for the cloning of mammalian cells after the success of other species (Cibell 2002, p. 32). Cloning has its basis on the understanding of the processes involved in ...
TY - JOUR. T1 - Molecular cloning of the cDNA for the catalytic subunit of human DNA polymerase δ. AU - Yang, Chun li. AU - Chang, Long sheng. AU - Zhang, Peng. AU - Hao, Huiling. AU - Zhu, Lingyun. AU - Lan Toomey, N.. AU - Lee, Marietta Y.W.t.. N1 - Funding Information: This work was supported by National Institutes of Health Grant GM31973 to MYWTL and in part by grants from the National Cancer Institute (CA 54323) and the Bremer and Milheim Foundation to LSC. An account of this work was presented at the Cold Spring Harbor Meeting on EukaryorJc DNA Replication in September 1991. We thank Drs A. Sugino, A. Morrison and G. Pignede for copies of their papers in press.. PY - 1992/2/25. Y1 - 1992/2/25. N2 - The cDNA of human DNA polymerase δ was cloned. The cDNA had a length of 3.5 kb and encoded a protein of 1107 amino acid residues with a calculated molecular mass of 124 kDa. Northern blot analysis showed that the cDNA hybridized to a mRNA of 3.4 kb. Monoclonal and polyclonal antibodies to the ...
A DNA fragment from Bacillus natto IFO3936 has been cloned which enhances the production of both extracellular alkaline and neutral proteases in Bacillus subtilis. The DNA sequence analysis around the gene responsible for the hyperproduction, prtR, revealed one open reading frame (comprising 60 amino acid residues) which was bounded by potential transcriptional and translational regulatory signals in its preceding and following regions. This open reading frame was not homologous to the published sequences of the structural genes of the two proteases. The calculated molecular weight (7,109) of the polypeptide predicted from the DNA sequence is much smaller than those of the two proteases, indicating that the gene product is distinct from those enzymes. In-frame fusion between the N-terminal region of the coding sequence and the lacZ gene of Escherichia coli demonstrated that the coding region was indeed translated in vivo. By deletion analysis it was suggested that prtR was the structural gene ...
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Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector back bone via side directed mutagenesis. Furthermore the original multiple cloning site was replaced for a multiple cloning site compatible to the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for in vitro applications the new multiple cloning site allows to express proteins with a Strep-tag II. Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in the yeast Saccharomyces cerevisiae. Moreover we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities. ...
Misc.Comments : Deposited by: Ichiro N. Maruyama Restriction digests of the clone give the following sizes (kb): HindIII--24.1, 17.6; EcoRI--22.0, 20.0; BamHI--24.8, 16.9; XbaI--32.7,9.0; NotI--41.7. (ATCC staff) Vector useful for constructing cDNA libraries. Permits positive selection for inserts using the Spi- phenotype, and excision of phagemid by lox/cre site-specific recombination. [1] To prepare phagemid from lambdaMGU2, grow recombinants on a RecA- host expressing the Cre protein (E. coli 1046[pCRE1], ATCC 77368) and select for ampicillin resistance. The pMGU product is 4.185 kb. [1] The order of the major features in the cloning region of the lambda vector is: lambda J - SmaI - SalI - loxP - EcoRI - M13 ori - ampR - pMB1 ori - HindIII - 3gam/BamHI/5gam - XhoI - loxP - SalI - lambda N. [1] Inserts can be amplified using the following primers flanking the BamHI cloning site: upstream 5-AAGAGGCAGAACTGGCAG-3 and downstream 5-ATCGATGCATAGCGATTC-3. [1] Efficiency of phagemid recovery is ...
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By Diego , source: Jan 31st, 2011 Well, unfortunately the weekend is over and the work week has begun. Today begins the five worst days of the week- the work days. At least you have us here at Daily Infographic to provide you with fun and interesting infographics. Todays infographic topic is cloning!. As long as I can remember there has been talk of cloning Personally I think cloning would be a pretty cool thing. I think it would be pretty cool to watch yourself, and even to interact with yourself. Hollywood has, of course, made many movies involving cloning. However, their cloning is as simple as entering a machine and there instantly being a copy of you. Science tells us that this is not in any way plausible and that cloning requires much more effort. If only everything were as simple as Hollywood makes it out to be.. As many people know, we really havent done that much in regards to cloning. In fact, we have only cloned a few animals and there hasnt even been a large of variety of animals ...
Introduction. Abbreviations and Acronyms. 1. FROM THE GENE TO THE TRANSGENIC ANIMAL.. Genome composition.. Gene structure.. The number of genes in genomes.. The major techniques of genetic engineering.. The systematic description of genomes.. Classical genetic selection.. Experimental mutation in genomes.. 2. TECHNIQUES FOR CLONING AND TRANSGENESIS.. Cloning.. Gene therapy.. Techniques of animal transgenesis.. 3. APPLICATIONS OF CLONING AND TRANSGENESIS.. Applications of animal cloning.. Applications of animal transgenesis.. 4. LIMITS AND RISKS OF CLONING, GENE THERAPY AND TRANSGENESIS.. Limits and risks of cloning.. Limits and risks of gene therapy.. Limits and risks of transgenesis.. Conclusion and Perspectives.. References.. Index. ...
Gene targeting is a powerful method that can be used for examining the functions of genes. Traditionally, the construction of knockout (KO) vectors requires an amplification step to obtain two homologous, large fragments of genomic DNA. Restriction enzymes that cut at unique recognitions sites and numerous cloning steps are then carried out; this is often a time-consuming and frustrating process. We have developed a one-step cloning method for the insertion of two arms into a KO vector using exonuclease III. We modified an adeno-associated virus KO shuttle vector (pTK-LoxP-NEO-AAV) to yield pAAV-LIC, which contained two cassettes at the two multiple-cloning sites. The vector was digested with EcoRV to give two fragments. The two homologous arms, which had an overlap of 16 bases with the ends of the vector fragments, were amplified by polymerase chain reaction. After purification, the four fragments were mixed and treated with exonuclease III, then transformed into Escherichia coli to obtain the desired
TY - JOUR. T1 - Structure of a family of rat amylase genes. AU - MacDonald, Raymond J.. AU - Crerar, Michael M.. AU - Swain, William F.. AU - Pictet, Raymond L.. AU - Thomas, Gilles. AU - Rutter, William J.. PY - 1980/12/1. Y1 - 1980/12/1. N2 - The sequences of two cloned rat pancreatic amylase cDNAs comprising 95% of the mRNA sequence are reported. Analysis of cloned rat genomic DNA fragments using cloned cDNA probes indicates that the rat genome contains multiple closely related amylase genes in which the cDNA sequences are distributed within a region 9 kilobases in length and are interrupted by at least seven intervening sequences.. AB - The sequences of two cloned rat pancreatic amylase cDNAs comprising 95% of the mRNA sequence are reported. Analysis of cloned rat genomic DNA fragments using cloned cDNA probes indicates that the rat genome contains multiple closely related amylase genes in which the cDNA sequences are distributed within a region 9 kilobases in length and are interrupted by ...
Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane. In brain, 2.0 kb mRNA was highly expressed. A Chinese case of X-linked acrogigantism and systemic review. Hong S, Kim TW, Choi I, Woo JM, Oh J, Park WJ, Kim DH, Cho C. Biochim Biophys Acta. Get the latest public health information from CDC: https://www.coronavirus.gov. While chromosome 19 only is the 19th largest autosomal chromosome, it contains 1440 protein-coding genes, and thus has the second highest number of protein-coding genes of any human chromosome. In brain, 2.0 kb mRNA was highly expressed. , M E Gåfvels, L G Paavola, C O Boyd, P M Nolan, F Wittmaack, A Chawla, M A Lazar, M Bucan, B O Angelin, J F Strauss, 3rd, Cloning of a complementary deoxyribonucleic acid encoding the murine homolog of the very low density lipoprotein/apolipoprotein-E receptor: expression pattern and assignment of ...
Scientists who do experimental genetics employ artificial selection experiments that permit the survival of organisms with user-defined phenotypes. Artificial selection is widely used in the field of microbial genetics, especially molecular cloning.. DNA recombination has been used to create gene replacements, deletions, insertions, inversions. Gene cloning and gene/protein tagging is also common. For gene replacements or deletions, usually a cassette encoding a drug-resistance gene is made by PCR.. Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of a single DNA molecule starting from a single living cell to generate a large population of cells containing identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the ...
This comprehensive yet balanced work emphasizes the principles and rationale underlying recombinant DNA methodology while furnishing a general understanding of the experimental protocols-suggesting flexible approaches to resolving particular molecular necessities that are easily adaptable to readers specific applications. Features summary tables presenting at-a-glance information on practices of recombinant DNA methodologies! Recombinant DNA Principles and Methodologies discusses basic and advanced topics requisite to the employment of recombinant DNA technology, such as plasmid biology nucleic acid biochemistry restriction enzymes cloning strategies gel electrophoresis southern and northern blotting preparation of probes phage lambda biology cosmids and genome analysis cloned gene expression polymerase chain reaction conventional and automated DNA sequencing site-directed mutagenesis and more! Elucidating the material with over 2250 edifying references, equations, drawings, and photographs, ...
Using a strategy based upon nucleotide sequence homology and starting from the sequence of the rat histamine H2 receptor (Ruat et al., Biochem. Biophys. Res. Commun. 1991, 179, 1470-1478), we have cloned a rat cDNA encoding a functional serotonin receptor (5-HT6). Its coding sequence corresponds to a glycoprotein of 436 amino acids displaying significant homology with other cloned monoaminergic receptors, e.g., various serotonin receptors. Genomic analysis of its gene indicated the presence of at least one intron. The major transcript of the 5-HT6 receptor gene has a size of approximately 4.1 kb but another minor 3.2 kb transcript was also evidenced. The highest expression, detected by Northern blot analysis as well as by in situ hybridization occurs in various serotoninergic areas of rat or guinea pig brain such as striatum, olfactory tubercle, nucleus accumbens and hippocampus, but a faint expression is also detectable in rat stomach. When transiently expressed in transfected COS-7 cells the 5-HT6
Cloning vectors A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating...
Polymerase chain reaction and rapid amplification of cDNA ends were used to isolate cDNAs encoding a 5-hydroxytryptamine3 (5-HT3) receptor subunit and its splice variants from guinea pig intestine. The amino acid sequence predicted from this cDNA is 81% homologous to the murine 5-HT3 receptor subunits cloned from NCB20 and N1E-115 cells. The splice variants code for two proteins differing by a deletion of six amino acids located in the large intracellular loop between transmembrane domains M3 and M4. For characterization, the cloned 5-HT3 cDNA was expressed in HEK 293 cells, and the electrophysiological and pharmacological properties of the recombinant ion/channel/receptor complex were investigated by patch clamping. Our data reveal that the cloned cDNAs code for guinea pig 5-HT3 receptors, which functionally assemble as homo-oligomers. The kinetic behavior of the ion channel and its sensitivity to several agonists and antagonists were markedly different from those of the cloned 5-HT3 receptors from
Abstract 【Aim】 This study aims to clone a C-type lectin gene from Plutella xylostella, to investigate its expression patterns and to elucidate its agglutination on bacteria. 【Methods】 Based on the bioinformatical analysis of genome and transcriptome database of P. xylostella, the full-length cDNA of a C-type lectin gene was cloned from P. xylostella by RT-PCR and rapid amplification of cDNA ends (RACE) techniques. Prokaryotic expression plasmid was constructed and the fusion protein was expressed in E.coli BL21. The polyclonal antibody with high serum titer was prepared using the purified fusion protein to immunize New Zealand white rabbit. Real-time quantitative PCR (RT-qPCR) was employed to analyze the expression profiles of this gene in different tissues (hemocyte, cuticle, fat body, midgut and Malpighiam tubules) of the day-1 4th instar larvae and different developmental stages (egg, 1st-4th instar larva, prepupa, pupa, and adult) of P. xylostella. RT-qPCR and Western blot were ...
The majority of chromosomal translocations breakpoints are within regions of the genome where few DNA probes are available. The use of yeast artificial chromosomes (YACs) containing long stretches of human DNA allows dispersed DNA markers to be used to identify the position of breakpoints but does not readily allow subcloning of the precise breakpoint within the YAC DNA nor the cDNAs containing the affected genes. We describe a procedure allowing rapid isolation of cDNAs corresponding to genes within a YAC clone. Random cDNA is hybridised to PCR-generated biotinylated fragments of total DNA from a yeast strain harbouring a YAC clone. The hybrids can be recovered to facilitate subsequent cloning of the cDNA molecules. The application of this method to the cloning of cDNA molecules carrying sequences involved in the translocation t(4;11)(q21;q23) is illustrated.
With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes. Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone
pUC18 is a commonly used plasmid cloning vector in E.coli. Due to a small size pUC18 enables successful cloning of large DNA fragments. Ampicillin resistant. High copy number. Blue/White colony screening.
TY - JOUR. T1 - Molecular cloning and characterization of oocyte-specific Pat1a in Rana rugosa frogs. AU - Nakamura, Yoriko. AU - Iwasaki, Takehiro. AU - Umei, Yosuke. AU - Saotome, Kazuhiro. AU - Nakajima, Yukiko. AU - Kitahara, Shoichi. AU - Uno, Yoshinobu. AU - Matsuda, Yoichi. AU - Oike, Akira. AU - Kodama, Maho. AU - Nakamura, Masahisa. PY - 2015/10/1. Y1 - 2015/10/1. N2 - The Pat1 gene is expressed in the immature oocytes of Xenopus, and is reportedly involved in regulating the translation of maternal mRNAs required for oocyte-maturation. However, it is still unknown when Pat1a first appears in the differentiating ovary of amphibians. To address this issue, we isolated the full-length Pat1a cDNA from the frog Rana rugosa and examined its expression in the differentiating ovary of this frog. Among eight different tissues examined, the Pat1a mRNA was detectable in only the ovary. When frozen sections from the ovaries of tadpoles at various stages of development were immunostained for Vasa-a ...
ID PMB preliminary; circular DNA; SYN; 5590 BP. XX AC ATCC77385; XX DT 03-FEB-1994 (Rel. 8, Created) DT 01-JUL-1995 (Rel. 12, Last updated, Version 1) XX DE Vertebrate/E.coli plasmid vector pMB - incomplete. XX KW cloning vector. XX OS Cloning vector OC Artificial sequences; Cloning vehicles. XX RN [1] RC pS1 from pUC19 & oligo RC pS1-polyA from pS1 & pMAMneo RC pS1-MT from pS1 & pT24 RC pS1-MMTV from pS1 & pMAMneo RC pS3 from pS1 RC pS5 from pS3 RC pK from pUC19 & oligo RC pM from pK & pS1-polyA & pS1-MMTV, RSV LTR/MMTV LTR RC pT from pK & pS1-polyA & pS1-MT, MT gene RC pM-hyg from pM & pHT, hyg gene RC pMB-hyg from pM-hyg & pS5, lacZ-amb RC pMB from pMB-hyg RC pT-hyg from pT & pHT, hyg gene RC pTB-hyg from pT-hyg & pS5, lacZ-amb RC pTB from pTB-hyg RA Wang Q., Maher V.M., McCormick J.J.; RT Mammalian expression vectors with modulatable promoters and two RT multiple cloning sites; RL Gene 119:155-161(1992). XX CC Deposited by: J. Justin McCormick CC The first site was designed for insertion ...
February 6, 2003. SOUTH HADLEY, Mass.-Bacteria do it. Yeasts do it. Even some snails, shrimp, and aphids do it. But wait, while all of these creatures reproduce asexually through cloning, creating an exact replica of themselves, the cloning of more complex species, such as humans, still seems unnatural to many of us. Is it simply a case of getting used to a new technology, the way most of us got used to the idea of test tube babies over the past two decades? Or will reproductive cloning of humans ultimately be deemed unethical?. Questions such as these about cloning and stem-cell research-both for disease treatment and prevention and for reproduction-will be the focus of a series of events this spring on the theme, The Political Embryo: Reconceiving Human Reproduction, presented by Mount Holyoke Colleges Harriet L. and Paul M. Weissman Center for Leadership. In addition to a wide-ranging discussion of cloning, the series will look at the ethical and legal issues surrounding new and developing ...
Introduction. 3845 ??? Cloning, is it man playing god? Cloning has become currently one of the hottest topics in society because of the laboratory-produced sheep Dolly, named after the country singer Dolly Patron. The birth of this little cloned sheep shocked society tremendously and created many controversial discussions. In the wake of this human cloning, religious leaders all over the world have condemned the action citing the reason that it is playing of god. However, we must understand that we at the Consortium that are involved in this effort are as human as anyone else. There is almost no difference between in-vitro fertilization (IVF) and cloning, other than the fact that the source of the genetic material is slightly different. Cloning is not playing of god, rather it is a salutary act which can fulfill human needs. ...read more. Middle. Looking back in science in the past, this distrust is totally understandable and logical. As an example, people think about atomic energy and above all ...
The risks of animal cloning are immense. The cloning process is inefficient and cloned animals have been observed to have higher rates of infection, tumour growth, and skeletal abnormalities than normal offspring. Are the risks and disadvantages of cloning because it is a nascent technology that scientists are trying to get to grips with, or are there inherent problems with the cloning process?
View Notes - unit7 cloning animals from BIOCHEM 100 at UMass (Amherst). UNIT 7 Animal Cloning and Epigenetics F08 The first cloned horse. What cell parts would you need to clone a human? (or