Plasmid DNA Extraction Using Alkaline Lysis Method

Plasmids can be isolated by a variety of methods many of which rely on the differential denaturation and reannealing of plasmid DNA compared to chromosomal DNA. One commonly used technique developed by Birnboim and Doly involves alkaline lysis. This method essentially relies on bacterial lysis by sodium hydroxide and sodium dodecyl sulfate (SDS), followed by neutralization with a high concentration of low-pH potassium acetate. This gives selective precipitation of the bacterial chromosomal DNA and other highmolecular-weight cellular components. The plasmid DNA remains in suspension and is precipitated with isopropanol.
Materials:

• Luria Bertani (LB) broth bacteria culture medium: 1% Tryptone, 0.5% yeast extract, 200 mM NaCl. Sterilize by autoclaving in suitable aliquots. In order to ensure retention of the plasmid, media should be supplemented with the appropriate antibiotic(s).
• 1.5 mL Microfuge tubes.
• Sterile tubes: Must have a volume of at least 10 mL to ensure good aeration.
• Lysis solution: 200 mM NaOH, 1% SDS. Store at room temperature.
• Resuspension solution: 50 mM glucose, 50 mM Tris-HCl, pH 8.0, 10 mM ethylene diamine tetraacetic acid (EDTA). Keep at 4^oC to prevent growth of contaminants.
• Potassium acetate (neutralizing solution): 3 M potassium/5 M acetate. For 100 mL, take 29.4 g of potassium acetate, add water to 88.5 mL, and 11.5 mL of glacial acetic acid. Store at room temperature.
• TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.
• Isopropanol.
• 70% Ethanol.

Methods:

• Take a number of separate sterile tubes and place 2 mL of L-broth into them. Inoculate from individual bacterial 37°C overnight with shaking.
• Transfer each culture to a labeled 1.5-mL Eppendorf tube, and centrifuge for 30 s at high speed in the microfuge.
  Here, a tight creamy pellet may be seen.
• Decant the supernatant and place tubes in a rack vertically for 5-10 s. Remove any of the remaining liquid by aspiration with a fine Pasteur pipet.
• Add 100 microliters of resuspension solution into each tube, close the lids, and resuspend the bacteria in each tube by shaking or vortexing to dissociate the bacterial pellet.
• To each tube add 200 microliters of lysis solution and mix by inverting the tube several times.
  The solution should quickly turn transparent and become more viscous indicating bacterial
  lysis has taken place.
• Allow at least 2-3 min for lysis to take place and leave the tubes to stand for 60 s before opening. This will allow the liquid to return to the bottom of the tube.
• To each tube add 150 microliters of neutralizing solution and invert the tubes several times. At this point bacterial chromosomal DNA is usually seen as a white precipitate.
• Centrifuge the tubes for 2-5 min at full speed in a microfuge.
• Place new sterile tubes into a rack, label them, and add 250 ~ of isopropanol to each tube.
• Remove the tubes from the microfuge, being careful not to disturb the precipitate.
• Remove the supernatant with a 1-mL pipet, avoiding the white precipitate as much as possible.
  Some of the precipitate may float, so it is critical to use a pipet and disposable tips to
  recover the supernatant rather than pouring it.
• Transfer the liquid phase into the new set of labeled tubes containing the isopropanol.
• Vortex the tubes for 5-10 s and centrifuge the tubes in the microfuge for 30 s at high speed. The plasmid DNA precipitates as a white pellet.
• Decant the supernatant and wash the pellets by adding 750 mL of 70% ethanol, vortex briefly, and centrifuge at high speed for 30 s.
• Decant the ethanol, and centrifuge again for 10 s to collect the remaining ethanol at the bottom of the tubes. Carefully aspirate the remaining ethanol and leave the tubes to air dry on the bench for 5 min.
• Dispense 50 microliter of TE into each tube, and resuspend the pellet
  It is not advisable to vortex, as this may lead to DNA shearing. The sample is best left for
  3-5 min with occasional finger flicking of the tube.
• Take 10 microliters of the resuspended pellet and analyze by agarose gel electrophoresis (see on my posting entitled Agarose Gel Electrophoresis of Nucleic Acids

Size Exclusion Chromatography

Size Exclusion Chromatography (also known as gel filtration) separates molecules based on molecular size. This chromatography can be applied using resins or membrane. With membranes, the smaller molecules pass through while the larger molecules (above a certain size cut-off) are held above the membrane. With resins, the larger molecules pass/flow through the resin and are collected first while the smaller molecules take longer to flow through because these smaller particles get held up within the pores of the resins. Therefore, with resins, the sample passes through the resin in decreasing molecular weight. Common size exclusion applications include concentration, fractionation, desalting and buffer exchange.  

Size exclusion is one of the easiest chromatography methods to perform because samples are processed using an isocratic elution. In its analytical form, size exclusion can distinguish between molecules (e.g. proteins) with a molecular weight difference of less than a factor of two times. In this application, the porosity of the filtration media to be used is selected to provide high resolution in the molecular weight range of interest.

How to Make and Run an Agarose Gel (DNA Electrophoresis)

Gel electrophoresis is a method that separates (based on size, electrical charge and other physical properties) macromolecules such as nucleic acids or proteins.
The electrophoresis term is used to describe the migration of charged particle under the influence of an electric field. Thus gel electrophoresis refers is the technique in which molecules are forced across a span of gel, motivated by an electrical current. On either end of the gel there are activated electrodes that provide the driving force. Therefore a molecule's properties especially the possession of ionisable groups, determine how rapidly an electric field can move the molecule through a gelatinous medium.
One very important application for gel electrophoresis is in DNA Technology. Biotechnology has for thousands of years been used by people who have used yeast to make flour into bread and grapes into wine. We are now using biotechnology to study the basic processes of life, diagnose illnesses, and develop new treatments for diseases. Some of the tools of biotechnology are natural components of cells. For example restriction enzymes are made by bacteria to protect themselves from viruses. They inactivate the viral DNA by cutting it in specific places. DNA ligase is an enzyme that exists in all cells and is responsible for joining together strands of DNA. Restriction enzymes can be used to cut DNA at specific sequences called recognition sites. They then rejoin the cut strands with DNA ligase to new combinations of genes. Recombinant DNA sequences contain genes from two or more organisms.
This technique has allowed researchers to gain the ability to diagnose diseases such as sickle cell anemia, cystic fibrosis, and Huntington's chorea early in the course of the disease. Many researchers are also applying the techniques of biotechnology to find new treatments for genetic diseases.
DNA technology has triggered research advances in almost all fields of biology. Currently hundreds of useful products are produced by genetic engineering. It has become routine to combine genes from different sources, usually different species--in test tubes, and then transfer this recombinant DNA into living cells where it can be replicated and expressed.
The most important achievements resulting from recombinant DNA technology have been  advances in our basic understanding of eukaryotic molecular biology. For example, only through the use of gene-splicing techniques have the details of eukaryotics gene arrangement and regulation been opened to experimental analysis.
Gel Electrophoresis is one of the staple tools in molecular biology and is of  critical value in many aspects of genetic manipulation and study. One use is the identification of particular DNA molecules by the band patterns they yield in gel electrophoresis after being cut with various restriction enzymes. Viral DNA, plasmid DNA, and particular segments of chromosomal DNA can all be identified in this way. Another use is the isolation and purification of individual fragments containing interesting genes, which can be recovered from the gel with full biological activity.
Gel electrophoresis makes it possible to determine the genetic difference and the evolutionary relationship among species of plants and animals. Using this technology it is possible to separate and identify protein molecules that differ by as little as a single amino acid.

Free Ebook Download Fundamentals of Biochemical Engineering

 

The biology, biotechnology, chemistry, pharmacy and chemical engineering students at various universtiy and engineering institutions are required to take the Biochemical Engineering course either as an elective or compulsory subject. This book is written keeping in mind the need for a text book on afore subject for students from both engineering and biology backgrounds. The main feature of this book is that it contains the solved problems, which help the students to understand the subject better. The book is divided into three sections: Enzyme mediated bioprocess, whole cell mediated bioprocess and the engineering principle in bioprocess.
DOWNLOAD HERE

Free Ebook Download Immunobiology 6th Edition

 

Charles A. Janeway, Paul Travers, Mark Walport and Mark Shlomchik, "Immunobiology"
Publisher: Garland Science | 2004 | ISBN: 0815341016 | File type: PDF | 910 pages | 18.4 mb

Immunobiology, Sixth Edition guides the reader through the immune system in all its aspects - from the first engagement of innate immunity to the generation of the adaptive immune response and its clinical consequences. The Sixth Edition has been thoroughly revised and updated, and now includes end-of-chapter questions. Immunobiology sets the standard for currency and authority with its clear writing style and organization, full-color art program, scientific accuracy, frequent updates, and consistent viewpoint - that of the host's interaction with an environment containing many species of potentially harmful microorganisms.
The Sixth Edition of Immunobiology includes a CD-ROM with original immunological animations based on figures in the book and videos selected from visually compelling experiments. All the animations and videos are accompanied by a voice-over narration. The CD also contains an archive of all the figures in the book, loaded into PowerPoint presentations. There is one presentation for each chapter of the book, and figures follow the order of the chapter.
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Combination of Gene Therapy and Chemotherapy Stops Kidney Cancer in Mouse Model

A novel therapeutic approach combining a modified viral vector and a small molecular weight drug produced promising results in a mouse model of human kidney cancer.

Investigators at the Virginia Commonwealth University (Richmond, USA) created a unique adenovirus vector by combining the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus. This Ad.5/3 adenovirus was then loaded with the gene needed to express the cancer-killing protein MDA-7/IL-24.

The viral vector was administered to mice bearing human renal carcinoma cells (RCCs), alone or together with the drug sorafenib, a small molecular weight inhibitor of several tyrosine protein kinases. Sorafenib, which is unique in targeting the Raf/Mek/Erk pathway (MAP Kinase pathway), has already been approved by the [US] Food and Drug Administration (FDA) for the treatment of renal carcinoma.

Results published in the December 15, 2010, issue of the journal Cancer Biology & Therapy revealed that infection with the Ad.5/3-mda-7 vector caused kidney cancer cells and normal cells lining the kidneys to secrete MDA-7/IL-24. MDA-7/IL-24 quickly stopped the growth of the primary tumor. As the infected cells continued to secrete MDA-7/IL-24, it entered the blood stream and eventually stopped the growth of a second, distinct tumor not directly infected by the adenovirus. Only renal carcinoma cells were destroyed by this “toxic bystander effect”; normal cells were unaffected. Sorafenib enhanced MDA-7/IL-24 toxicity and significantly increased its antitumor effects in the mouse model.

“While further research is needed, this therapy could be a novel and effective way to treat metastatic kidney cancer and prolong patient survival,” said senior author Dr. Paul Dent, professor of biochemistry at Virginia Commonwealth University. “This is the first study to clearly define that gene therapeutic delivery of MDA-7/IL-24 in kidney cancer should be explored in the clinic, especially since we have demonstrated an established, FDA-approved drug enhances its toxicity to cancer cells.”

Cell Culture Technique (Attached Cell)