reagent NB: • Using PLUS™ Reagent (Cat. No. 11514-015) enhances transfection performance in HUVEC cells. • The addition of antibiotics to media during transfection may result in cell death Transfection of HMECs Use this procedure to transfect plasmid DNA into HMECs cells in a 12-well format All amounts and volumes are given on a per well basis. 1. Cell density should be 50~80% confluent on the day of transfection (use the normal growth medium without antibiotics). 2. For each well of cells
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Report Backround: The plasmid pGLO contains an antibiotic-resistance gene‚ ampR‚ and the GFP gene is regulated by the control region of the ara operon. Ampicillin is an antibiotic that kills E. coli‚ so if E. coli‚ so if E. coli cells contain the ampicillin-resistance gene‚ the cells can survive exposure to ampicillin since the ampicillin-resistance gene encodes an enzyme that inactivates the antibiotic. Thus‚ transformed E. coli cells containing ampicillin-resistance plasmids can easily be selected
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process which involves plasmid DNA being bound to the cell surface and the subsequent uptake of DNA by the cell (Panja et al.‚ 2008). For artificial transformation of E. coli cells with plasmids‚ plasmid DNA has to be extracted from bacterial cells using the High-Speed Plasmid Mini Kit‚ which is then mixed with competent E. coli cells followed by heat shock and the streaking of transformed cells on two different types of agar plate (LB and LB+ampicillin). The extracted plasmid DNA is important as it
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Detection of plasmids in arsenic (As) resistant bacteria isolated from As-contaminated groundwater Mahima Rani and Pinaki Sar* Department of Biotechnology‚ Indian Institute of Technology‚ Kharagpur‚ West Bengal–721302‚ India mahima06bt11@gmail.com‚ Ph: +91 8348523016 Abstract Role of plasmids in conferring resistance to several heavy metals and antibiotics to naturally occurring bacteria is well known. In contaminated environments‚ presence of metal resistant genes on plasmids often provide
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of this experiment‚ we transformed the bacteria into an antibiotic resistant form by inserting a plasmid into it. We used heat shock in order to make the bacteria capable to uptake a plasmid in the presence of calcium ions that help disrupt the cell membrane (heat shock is the combination of altering hot and cold). When they are capable of accepting plasmids‚ the bacteria are incubated with plasmids that carry the resistance to a particular antibiotic‚ in this case ampicilin. We also ran a control
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uses of plasmids in G.M. experimentation. Plasmids are extrachromosomal genetic elements found in a variety of bacterial species. They are double stranded; autonomously replicating‚ supercoiled‚ covalently closed circular (ccc) DNA molecules that range in size from 1 kb to greater than 200 kb. Often‚ plasmids contain genes coding for enzymes that‚ under certain circumstances‚ are advantageous to the bacterial host (Table 1). Table 1. Some of the phenotypes conferred by different plasmids that
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Material and methods Hosts ‚ plasmids and chemicals E. coli TOP10 strain was used for cloning and proliferation. E.coli BL21 DE3 was used as expression host cell. The pET 28a(+) plasmid was employed for gene expression. All chemicals were obtained from Merck Company (Germany). Codon optimization and gene synthesis Sequence encoding V-domain was obtained from Swiss-port‚ Uniprot KB and National Center for Biotechnology Information (NCBI) databases. 6x His-tag sequence was placed at N-terminal
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Title: E. Coli Transformation with a Plasmid DNA Containing the GFP Gene Introduction: Bacterial transformation is the process of bacteria taking in and expressing exogenous DNA. This has led to many other discoveries. In order for bacterial transformation to occur the bacteria must be in a certain physical state to be able to take in DNA. This is called competency and it allows the cell membrane to be permeable so DNA can pass through. Currently researchers are studying the transformation
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buffer (pH 6‚0) overnight at room temperature‚ frozen in liquid nitrogen and stored at -80 ˚C after 0.22 μm pore membrane filtration. Evaluation of DNA-protein interaction by gel retardation assay Protein-DNA complexes were formed using 1 µg of plasmid pTriEx-1.1 Hygro at different pDNA: protein molar ratios (1:100‚ 1:200‚ 1:500‚ 1:1000‚ 1:2000‚ 1:8000) during 10 minutes. The increasing molar ratios were assessed by gel retardation assay on a 0.8% agarose gel and visualized by ethidium bromide staining
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I. Title Identification of an Unknown Plasmid In this experiment‚ we determined the phenotypic capability of an unknown plasmid along with its size. With the use of gel electrophoresis‚ we analyzed the gel photograph by using a standard DNA marker‚ Lambda HindIII‚ and came to a conclusion based on our results. II. Abstract Two experiments were done to identify an unknown plasmid. The success of these experiments came from the use of modern day technology involving gel electrophoresis
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