Book of moon pgl2 basic plasmid

Each plasmid was transfected into vsmc using superfect reagent qiagen according to the manufacturers instructions 29. Demethylation of the coding region triggers the activation. Potential enhancer elements can also be inserted upstream of the promoter. This is a free resource for the scientific community that is compiled by addgene. This resource is designed to educate all levels of scientists and plasmid lovers and serves as an introduction to plasmids, allowing you to spend less time researching plasmid basic features and spend. Nutritional regulation of the fatty acid synthase promoter. The size of the pcr products was confirmed by electrophoresis and by dna sequencing. Addgene is a nonprofit plasmid repository dedicated to improving life science research. Characterization of the human edf1 minimal promoter.

Suppression of vascular smooth muscle cell responses. Numerous configurations of pgl4 vectors are available, including those with the synthetic firefly luc2 photinus pyralis and. Randall moon lab plasmids create a link to this page the randall moon lab has deposited plasmids at addgene for distribution to the research community. Gene in human breast carcinoma cells is mediated via an imperfect halfpalindromic estrogen response element and sp1 motifs. Transgenic mice harboring the cat gene driven by the various 59 deletions of the fas promoter were generated, and the transgenic mice were identified by pcr of the tail dna as described 9, 26. The pgl4 luciferase reporter vectorsa,b,c,d are the next generation of reporter gene vectors optimized for expression in mammalian cells. The plasmids, pgl2 sv40 and pgl2 basic were used as positive and negative controls, respectively. The pgl2basic vector lacks eukaryotic promoter and enhancer sequences, allowing maximum flexibility in cloning putative regulatory sequences. Analysis of pgl2 182 f1 and pgl2 697 f2 reporter plasmids gave similar luciferase activity in arh77 and hela cells, supportive of.

A survey of molecular cloning vectors and their uses focuses on the functions of molecular cloning vectors. Construction and identification of recombinant firefly. Therefore, expression of zta is a prerequisite for activation of the ebv lytic cycle. Expression of luciferase activity in cells transfected with this plasmid depends on insertion and proper orientation of a functional promoter upstream from luc. Plasmid construction the pref1 6000luc plasmid was generated by first ligating an. For example, a variety of highcopy number yeast vectors have been derived from the natural yeast 2micron plasmid. The pgl4 luciferase reporter vectors are the next generation of reporter gene vectors optimized for more efficient expression in mammalian cells. A laboratory manual is a singlevolume adaptation of the threevolume third edition of molecular cloning.

Promoterless vector for measuring the activity of promoter and enhancer sequences with a luciferase assay. Cdk2 mrna capsite inserted into the pgl2basic plasmid promega. These cards are for sale individually to see the price for each card please select the specific card in the drop down menu above. Wisp1 is a wnt1 and bcatenin responsive oncogene lifeng xu, 1ryan b. Te buffer is commonly used to redissolve dna because it. The ppdha2topo recombinant was also digested with enzymes mslixhoi and the 117 bp fragment was subcloned in the pgl2 reporter plasmid generating the. In pgl2based recombinant plasmids, the origin of replication can be eliminated in. Our long term goal is to evaluate the use of rapidly internalizing antibody to selectively deliver radiolabeled dna to the nucleus. Written by acknowledged experts in the field, this volume provides an up to date treatment of the structure, function and application of plasmids with a particular emphasis on current and future trends. The size of the pcr products was confirmed by electrophoresis and dna sequencing. Ijms free fulltext plasmids from food lactic acid bacteria. The plasmid pgl2 replicates via the rolling circle mechanism, however, the. Plasmid construction and production of transgenic mice.

The pgl4 luciferase reporter vectorsae are the next generation of reporter gene vectors optimized for expression in mammalian cells. Estradiol regulation of the human retinoic acid receptor. And bought this structure deck and as a whole its a solid deck but it comes with a bunch of individual components that really shine, for example dark law, shadow mist need 3 comes with 1 bubble man, summoner monk, mask change need 3 comes with 1 a hero lives comes with 1 need 23 all these are really solid staples in a hero deck no. In nature, the two main routes of plasmid transfer are conjugation and natural transformation. Accordingly, the various pdha2 reporter plasmids and the parental pgl2 basic vector were transfected into hela.

Restriction enzymes and nucleotide sequence conformed that the coupling site of recombinant plasmid was correct without base mutation and deletion, and the sequence inserted was the same as data of genebank. Overseas customers please use paypal or contact me to arrange the payment. Uk customers can pay by paypal, cheque or postal order. Here we have for sale individually 91 different cards from premium gold. We used a minimum of two independent founder lines for the. After isolation from the mg1655 pgl2 atp strain, the recombinant pgl2 atp plasmid carrying the. Recently, a novel plasmid pmc11 was isolated from l. Pipet the 20 ul in and out, up the side of the tube to ensure that all of the plasmid dna comes into contact with the te buffer. When the ethanol is gone you can check this by smelling the tube add 20 ul of te buffer 10 mm trishcl, 1 mm edta, ph 8. Pc12 cells were cotransfected with the firefly luciferaseexpressing plasmid faslluc or pgl2basic together with the renilla luciferaseexpressing plasmid prltk or phrgb and 20 or 40 ng expression vector. Levine1,3,4 1department of molecular biology, princeton university, princeton, new jersey 08544 usa. Cloning of the mouse pax4 gene promoter and identification.

Topics include criteria for plasmid vector design, construction and structure, transcriptional signals, dna replication, recombination, mobilization, and plasmid stability. When compared to pgl2 basic vector, pgl2 01 gave a 100fold activation of the luciferase activity, indicating the presence of promoter activity. A novel approach for escherichia coli genome editing. However, it contains a multiple cloning region to allow cloning of a promoter of choice. The genomic structure and promoter analysis of the human. Nurr1 affects prltk but not phrgb internal control. In the lab, we use chemical transformation, electroporation, and transfection to put plasmids into cells. Utr, were inserted in front of the firefly luciferase reporter gene. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources.

For the increasing number of professionals needing to learn about and use molecular biology techniques in their research, the daunting challenge is to break through the technical and largely inaccessible jargon found in most texts. Human tumor necrosis factor receptor p7580 cd120b gene structure and promoter characterization sybil m. Use text editor or plasmid mapping software to view sequence. Demethylation of the coding region triggers the activation of the. Pdf estradiol regulation of the human retinoic acid. A novel approach for escherichia coli genome editing combining in. Plasmids pglpromoter and pgl2 basic were purchased from promega.

Inhibitory effect of esculetin on migration, invasion and. The pgl4 vectors are engineered with fewer consensus regulatory sequences and a synthetic gene, which has been codon optimized for mammalian expression. Human tumor necrosis factor receptor p7580 cd120b gene. We have recently shown that nuclear import of plasmid dna is a sequencespecific event, requiring the sv40 enhancer, a region known to bind to a number of general transcription factors. The book is aimed primarily at research scientists, graduate students and professional scientists but will also be of great interest to all molecular biologists and microbiologists. After i make the plasmid then i can follow some protocol to do the transformation. The pgl2 basic vector containing a polyadenylation signal upstream of the luciferase gene was used to construct the expression vectors by subcloning pcramplified dna of the mmp9 promoter into the sacihindiii site of the pgl2 basic vector 15. Search the worlds most comprehensive index of fulltext books. Role of rta in the translation of bicistronic bzlf1 of. Mutation of a nuclear respiratory factor 2 binding site in. To address this limitation, we have begun to develop cellspecific vectors whose specificity is at the level of the nuclear import of the plasmid dna. A cytokine responsive construct, pgl2saa2pt, was generated by cloning the acute phase promoter of human serum amyloid a2 saa2 upstream of a luciferase reporter gene. Serial deletion constructs of the zic3 promoter were subcloned into pgl2basic plasmid. As a dna sequence we used the pgl2 plasmid containing the luciferase reported gene.

Basic and the sv40luciferase pgl2promoter vector were purchased from. As a model system we used t101, an igg2a murine moab that recognizes cd5 and upon binding to this antigen rapidly internalizes. Welcome to restrictionmapper on line restriction mapping the easy way. Relative to pgl2 basic alone, a higher luciferase activity was obtained for pgl2 182 f1 and pgl2 697 f2 in all cell lines tested, demonstrating the analyzed regions indeed contained a functional promoter.

We will transform this plasmid, which carries a gene encoding a protein which destroys the antibiotic ampicillin into bacterial cells which. In the first part of todays lab we will demonstrate that the dna which we extracted last week can create heritable changes in the phenotype of bacterial cells. Zic3 promoterluc plasmid dnas were injected at 20 pg per embryo into. A desktop resource, is to curate a onestop reference guide for plasmids. Among the crucial basic aspects of chromosomal insertion techniques. Randall moon s lab contains the insert betacateninmyc.