Monolithic Columns for Purification and In-Process Control of Viruses and Virus-Like Particles" in "Vaccine Technology IV", B
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Agarose Gel Electrophoresis
Laboratory for Environmental Pathogen Research Department of Environmental Sciences University of Toledo Agarose gel electrophoresis Background information Agarose gel electrophoresis of DNA is used to determine the presence and distinguish the type of nucleic acids obtained after extraction and to analyze restriction digestion products. Desired DNA fragments can be physically isolated for various purposes such as sequencing, probe preparation, or for cloning fragments into other vectors. Both agarose and polyacrylamide gels are used for DNA analysis. Agarose gels are usually run to size larger fragments (greater than 200 bp) and polyacrylamide gels are run to size fragments less than 200 bp. Typically agarose gels are used for most purposes and polyacrylamide gels are used when small fragments, such as digests of 16S rRNA genes, are being distinguished. There are also specialty agaroses made by FMC (e.g., Metaphor) for separating small fragments. Regular agarose gels may range in concentration from 0.6 to 3.0%. Pouring gels at less or greater than these percentages presents handling problems (e.g., 0.4% agarose for genomic DNA partial digests requires a layer of supporting 0.8% gel). For normal samples make agarose gels at 0.7%. The chart below illustrates the optimal concentrations for fragment size separation. The values listed are approximate and can vary depending on the reference that is used. If you do not know your fragment sizes then the best approach is to start with a 0.7% gel and change subsequently if the desired separation is not achieved. Nucleic acids must be stained prior to visualization. Most laboratories use ethidium bromide but other stains (e.g., SYBR green, GelStar) are available. -
Development and Optimization of Organic Based Monoliths for Use in Affinity Chromatography
University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Student Research Projects, Dissertations, and Theses - Chemistry Department Chemistry, Department of Winter 12-2-2011 Development and Optimization of Organic Based Monoliths for Use in Affinity Chromatography Erika L. Pfaunmiller University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/chemistrydiss Part of the Analytical Chemistry Commons Pfaunmiller, Erika L., "Development and Optimization of Organic Based Monoliths for Use in Affinity Chromatography" (2011). Student Research Projects, Dissertations, and Theses - Chemistry Department. 28. https://digitalcommons.unl.edu/chemistrydiss/28 This Article is brought to you for free and open access by the Chemistry, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Student Research Projects, Dissertations, and Theses - Chemistry Department by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. DEVELOPMENT AND OPTIMIZATION OF ORGANIC BASED MONOLITHS FOR USE IN AFFINITY CHROMATOGRAPHY by Erika L. Pfaunmiller A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfilment of Requirements For the Degree of Master of Science Major: Chemistry Under the Supervision of Professor David S. Hage Lincoln, Nebraska December, 2011 DEVELOPMENT AND OPTIMIZATION OF ORGANIC BASED MONOLITHS FOR USE IN AFFINITY CHROMATOGRAPHY Erika L. Pfaunmiller, M.S. University of Nebraska, 2011 Adviser: David S. Hage Affinity chromatography is an important and useful tool for studying biological interactions, such as the binding of an antibody with an antigen. Monolithic supports offer many advantages over traditional packed bed supports in affinity chromatography, including their ease of preparation, low back pressures and good mass transfer properties. -
Purification of Recombinant Vaccinia Virus for Oncolytic and Immunotherapeutic Applications Using Monolithic Column Technology
Purification of Recombinant Vaccinia Virus for Oncolytic and Immunotherapeutic Applications using Monolithic Column Technology A thesis submitted to University College London for the degree of Doctor of Engineering by David Isaac William Vincent The Advanced Centre for Biochemical Engineering Department of Biochemical Engineering University College London Gordon Street London WC1H 0AH 1 I, David Vincent confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed……………….………………………Date……………………………………… 2 Acknowledgements I would like to thank my supervisor Dr Yuhong Zhou for her invaluable feedback and support over the course of this EngD. Without her continuous encouragement this work would likely never have been completed. I would also like to thank my Advisor Prof. Ajoy Velayudhan for his help and guidance. Many a long discussion was had on the intricacies of vaccinia viral purification which were thought provoking, inspirational but also really good fun. I would also like to express my gratitude to the staff and students at UCL. There are too many to mention everyone, but in particular I would like to thank Mrs Ludmila Ruban, Miss Dhushy Stanislaus and Dr Tarit Mukhopadhyay for their support, especially in setting up the vaccine lab in the department which I could benefit from in my final year. I would also like to thank Dr Patricia Perez Esteban for her support, friendship and bioprocess wisdom. I would like to express my gratitude to BIA Separations and the EPSRC for sponsoring this work. Without this financial support this work would not have been possible. -
Biomems Literature by Year Prof
BioMEMS Literature by Year Prof. Steven S. Saliterman 1. Xu M, Obodo D, Yadavalli VK. The design, fabrication, and applications of flexible bio- sensing devices. Biosensors & Bioelectronics. 2019;124:96-114. 2. Wongkaew N, Simsek M, Griesche C, Baeumner AJ. Functional Nanomaterials and Nanostructures Enhancing Electrochemical Biosensors and Lab-on-a-Chip Performances: Recent Progress, Applications, and Future Perspective. Chemical Reviews. 2019;119(1):120-194. 3. Wang MH, Yin HS, Zhou YL, et al. Photoelectrochemical biosensor for microRNA detec- tion based on a MoS2/g-C3N4/black TiO2 heterojunction with Histostar@AuNPs for signal amplification. Biosensors & Bioelectronics. 2019;128:137-143. 4. Wang JS, Hui N. Electrochemical functionalization of polypyrrole nanowires for the de- velopment of ultrasensitive biosensors for detecting microRNA. Sensors and Actuators B-Chemical. 2019;281:478-485. 5. Sun EWL, Martin AM, Young RL, Keating DJ. The Regulation of Peripheral Metabolism by Gut-Derived Hormones. Frontiers in Endocrinology. 2019;9. 6. Soler M, Huertas CS, Lechuga LM. Label-free plasmonic biosensors for point-of-care di- agnostics: a review. Expert Review of Molecular Diagnostics. 2019;19(1):71-81. 7. Soler M, Huertas CS, Lechuga LM. Label-free plasmonic biosensors for point-of-care di- agnostics: a review. Expert Review of Molecular Diagnostics. 2019;19(1):71-81. 8. Sola L, Damin F, Chiari M. Array of multifunctional polymers for localized immobilization of biomolecules on microarray substrates. Analytica Chimica Acta. 2019;1047:188-196. 9. Seidi S, Ranjbar MH, Baharfar M, Shanehsaz M, Tajik M. A promising design of microflu- idic electromembrane extraction coupled with sensitive colorimetric detection for col- orless compounds based on quantum dots fluorescence. -
BIA Separations CIM Monolithic Columns for Purification and Analytics of Biomolecules
BIA Separations CIM Monolithic Columns for Purification and Analytics of Biomolecules Lidija Urbas, PhD [email protected] Outline • BIA Separations • Chromatography – Monolithic Chromatography – Design of monolithic columns • DSP applications • PAT columns and applications: – Case study: combining DSP and PAT of Adenoviruses • Conclusions Ajdovščina BIA Separations • BIA Separations was founded in September 1998. • Headquarters in Austria, R&D and Production in Slovenia. BIA Separations • BIA Separations was founded in September 1998. • Headquarters in Austria, R&D and Production in Slovenia. • BIA Separations USA established in September 2007 - sales and tech support office. • BIA Separations China established in January 2011 - sales and tech support office. • 90 employees world wide • Main focus: To develop and sell methacrylate monolithic columns & develop methods and processes for large biomolecules separation and purification. BIA Separations – Products and Services CIM Contract monolithic Research columns Laboratory Method development and Technical Support Important Milestones • 2004: First monolith used for the industrial cGMP purification for plasmid DNA at Boehringer Ingelheim provide 15-fold increase in productivity • 2006: First cGMP production of a vaccine (influenza) using CIM® • 2008: OEM Partnership with Agilent Technologies – develop and produce analytical monolithic columns for PAT • In 2011 BIA Separations was awarded by KAPPA-Health as a model SME in the EU Co-funded research projects • 2012: co-marketing and -
Product Specifications Agarose Hires Molecular Biology Grade
Product Specifications Electrophoresis Reagents, Buffers, Agarose, Polymerase Chain Reaction Custom Primers and Probes Hybridization and Detection Reagents Agarose HiRes Molecular Biology Grade Store at Room Temperature Catalog Number Description Size 40-3015-10 Agarose HiRes Ultra Pure Molecular Biology Grade 100 gms 40-3015-50 Agarose HiRes Ultra Pure Molecular Biology Grade 500 gms 40-3015-01 Agarose HiRes Ultra Pure Molecular Biology Grade 1 KG Product Description & Application Agarose HiRes is certified Ultra Pure molecular biology grade DNase and RNase-free agarose powder. It is specifically recommended for resolution of short fragments ranging in size between 20 bp and 800 bp and is an excellent substitute for polyacrylamide electrophoresis for resolution of short DNA fragments. HiRes Agarose is commonly used for electrophoretic resolution of fragments obtained from amplification of short tandem repeats (STR’s), di, tri and tetra-nucleotide repeats, and other polymorphic loci. Specifications Appearance White homegeous powder Gel strength of 1.5 % (w/v) gel >1680g / cm 2 Gel strength of 3 % (w/v) gel >3290g / cm 2 Gelling temperature 33-34°C Melting temperature 74°C EEO: 0.1-0.2 Moisture: <4% DNase and RNase None detected High Resolution Gel Electrophoresis of DNA Gene Link HiRes agarose is an intermediate melting temperature agarose (~74°C) that provides one of the finest resolutions for DNA fragments from STR, tri and tetra-nucleotide repeat amplification and other length based polymorphisms. Using a 2 – 4% gel (made in either TAE or TBE) it is possible to resolve fragments that are anywhere from 20 – 800 bp in length. A 4% HiRes agarose gel can differentiate a 99bp fragment from a 110 bp fragment running the gels at 45 mAmps at room temperature. -
Agarose Gels (Horizontal Gel Electrophoresis)
TECHNIQUES IN MOLECULAR BIOLOGY – AGAROSE GELS (HORIZONTAL GEL ELECTROPHORESIS) DNA gels are used to separate fragments of DNA and RNA. Unlike most protein separations which use acrylamide polymers, use agarose in a submerged horizontal orientation, and at time called horizontal gel electrophoresis. This handout will cover the details of agarose gels, the theory of separation by agarose gel electrophoresis and tips for conducting successful gel electrophoresis. The basic principle of separation for all electrophoresis is the movement of a charged molecule in a medium subjected to an electric field. v=Eq/f V is the velocity of the molecule subjected to electrophoresis. E is the electrical field in volts/cm, q is the net charge on the molecule and f is the frictional coefficient. The impact of f depends on the mass and shape of the molecule. This equation simply explains that the rate (v) of a particle depends on the electrical field and charge but inversely impacted by the counteracting force generated by the viscous drag. Factors influencing F is of course the size and shape of the molecule. Think of a short linear oligonucleotide vs a large supercoiled plasmid vs long chromosomal DNA. Adding a value to f is the media through which the molecules migrate. Agarose is a seaweed extract (red algae agar) and is a long polymer of D and L galactose and derivatives in a linear polymer bonded by two different glycosidic bonds. Once hydrated and formed into a gel, the carbohydrate will form helical Repeating pattern of agarose fibers and aggregates creating channels of 50 to more than 200 nm in diameter. -
GENERAL BIOLOGY LABORATORY II Bioassays of Major Biomolecules: Nucleic Acids
Weeks 9-10: Bioassays of major biomolecules: Nucleic acids GENERAL BIOLOGY LABORATORY II Canbolat Gürses, Hongling Yuan, Samet Kocabay, Hikmet Geckil Department of Molecular Biology and Genetics Inonu University Weeks 9-10 Bioassays of major biomolecules: Nucleic acids DNA is the genetic material in all organisms. Scientists work with DNA for a variety of reasons, such as cloning, amplification, sequencing, and other genetic manipulations. In general, the first steps in DNA (or RNA) studies involve DNA isolation and their qualitative, and quantitative determination. DNA or RNA concentration in solution can be determined through the optic properties (max. absorbance) of nucleotides at 260 nm. Once their concentration and purity are determined, nucleic acids can be investigated with more specific and sensitive methods (e.g., agarose gel electrophoresis, etc.). 1 DNA can be extracted and isolated from any cell, tissue, or organ using a variety of methods such as alkali lysis, enzymatic lysis and boiling methods and it can be precipitated from the rest of cell components by ethanol, isopropanol precipitation. As we have seen for proteins which have specific absorbance maxima at 280 nm, nucleic acids absorb light maximally at 260 nm. However, while one unit A at 280 nm is equal to one unit of protein (as mg ml-1), for DNA and RNA 1 A unit at 260 nm is equal to about 50 and 40 µg ml-1, respectively. The cell extract is a mixture of all cell components and organelles. Once large particles (e.g., organelles, membrane fragments, etc) are removed by a low speed centrifugation, the solution part (i.e., supernatant) contains cell components such as proteins and nucleic acids dissolved in an aqueous environment. -
DNA Molecular Weight Marker VIII (19 – 1114 Bp) Pucbm21 DNA × Hpa II* Digested Pucbm21 DNA × Dra I* + Hind III* Digested
For life science research only. Not for use in diagnostic procedures. DNA Molecular Weight Marker VIII (19 – 1114 bp) pUCBM21 DNA × Hpa II* digested pUCBM21 DNA × Dra I* + Hind III* digested ־ Cat. No. 11 336 045 001 50 g 1 A260 unit y Version 19 50 g (200 l) for 50 gel lanes Content version: September 2018 Store at Ϫ15 to Ϫ25°C 1. Product Overview Content Ready-to-use solution in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 Concentration 250 g/ml Size Distribution Fragment mixture prepared by cleavage of pUCBM21 DNA with restriction endonucleases Hpa II * and Dra I* plus Hind III*. The mixture contains 18 DNA fragments with the following base pair lengths (1 base pair = 660 daltons): 1114, 900, 692, 501, 489, 404, 320, 242, 190, 147, 124, 110, 67, 37, 34, (2×), 26, 19 (determined by computer analysis of the pUCBM21 sequence). Application Molecular weight marker for the size determination of DNA in agarose gels. Properties After gel electrophoresis of 1 g of the fragment mixture in a 2% Aga- rose MP* gel 13 bands are visible. The 501 bp and 489 bp fragments as well as the fragments 37–19 bp run as one band. Typical Analysis Fig. 1: Separation of 1 g DNA Molecular Weight Marker VIII on a The DNA fragment mixture shows the typical pattern of 13 bands in 1.8% agarose gel. Ethidiumbromide stain. agarose gel electrophoresis.. 2. Supplementary Information Storage/Stability The unopened vial is stable at Ϫ15 to Ϫ25°C until the expiration date Changes to Previous Version printed on the label. -
Simian Adenovirus Vector Production for Early-Phase Clinical Trials: A
Vaccine 37 (2019) 6951–6961 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components Sofiya Fedosyuk a, Thomas Merritt b, Marco Polo Peralta-Alvarez a, Susan J Morris a, Ada Lam c, Nicolas Laroudie d, Anilkumar Kangokar c, Daniel Wright a, George M Warimwe e,f, Phillip Angell-Manning b, ⇑ Adam J Ritchie a, Sarah C Gilbert a, Alex Xenopoulos g, Anissa Boumlic d, Alexander D Douglas a, a Jenner Institute, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK b Clinical Biomanufacturing Facility, University of Oxford, Roosevelt Drive, Oxford OX3 7JT, UK c Millipore (UK) Ltd. Bedfont Cross, Stanwell Road, TW14 8NX Feltham, UK d Millipore SAS, 39 Route Industrielle de la Hardt, Molsheim 67120, France e Centre for Tropical Medicine and Global Health, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK f KEMRI-Wellcome Trust Research Programme, P.O. 230-80108 Kilifi, Kenya g EMD Millipore Corporation, 80 Ashby Road, Bedford, MA 01730, USA article info abstract Article history: A variety of Good Manufacturing Practice (GMP) compliant processes have been reported for production Available online 30 April 2019 of non-replicating adenovirus vectors, but important challenges remain. Most clinical development of adenovirus vectors now uses simian adenoviruses or rare human serotypes, whereas reported manufac- Keywords: turing processes mainly use serotypes such as AdHu5 which are of questionable relevance for clinical Simian adenovirus vaccine development. Many clinically relevant vaccine transgenes interfere with adenovirus replication, GMP whereas most reported process development uses selected antigens or even model transgenes such as Clinical trials fluorescent proteins which cause little such interference. -
DNA Molecular Weight Marker XIV (100–1500 Bp) 100 Base Pair Ladder
For life science research only. Not for use in diagnostic procedures. DNA Molecular Weight Marker XIV (100–1500 bp) 100 base pair ladder Cat. No. 11 721 933 001 y Version 08 c 50 g 1 A260 unit Content version: October 2018 50 g (200 l) for 50 gel lanes Store at Ϫ15 to Ϫ25°C Product overview Formulation Solution in 10 mM Tris-HCl, 1 mM EDTA, pH 8 Concentration 250 µg/ml Size distribution Fragment mixture prepared by cleavage of a specially constructed plasmid with restriction endonucleases. The mixture contains 15 double stranded DNA frag- ments with the following base pair lengths: 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 and an additional band of 2642 bp. The 500 bp and 1000 bp banding pattern is two to three times brighter. Application The DNA Molecular Weight Marker XIV allows accurate sizing of DNA fragments generated by PCR or restric- tion digest separated on agarose gels. The DNA molec- ular weight marker can be used in conjunction with DNA Molecular Weight Marker XIII (50 bp ladder)* for precise size determination. Electrophoretic separation of this molecular weight marker results in a regular pattern. The fragments have 5'-protruding ends which can be labeled by filling in reactions using e.g. [32P]-dTTP and [32P]-dGTP, or using DIG-11-ddUTP* and Terminal Transferase*. Typical analysis The DNA fragment mixture shows the ladder with 15 bands and an additional band of 2642 bp in agarose gel electrophoresis as shown in the figure. Fig.: Separation of 1 µg DNA molecular weight marker XIV on a 2% Separation Apply 1 µg DNA per lane on a 2% Agarose MP* gel. -
Section IV: Detection and Sizing of DNA in Agarose Gels
Section IV: Detection and Sizing of DNA in Agarose Gels In This Section Guide to Lonza Ladders and Markers 96 Detecting DNA with GelStar® and SYBR® Green Nucleic Acid Gel Stains 98 Detecting DNA with Ethidium Bromide 102 High Sensitivity Detection using the FlashGel® System 103 Sizing and Detection References 104 Gels in Agarose DNA of 95 1-800-638-8174 www.lonza.com/research Return to Main Section IV: Detection and Sizing of DNA in Agarose Gels Guide to Lonza Ladders and Markers Size Range (Bold numbers indicate brighter bands) 100 bp Quant Rev Quant 50 bp- 50 bp- 1 kb- 20 bp 20 bp Ext 100 bp ext Tandem 500 bp Ladder Ladder 1000 bp 2500 bp 10 kb Standard Ladders (Cat. No.) 50330 50320 50321 50322 NA 50323 50334 50335 50461 50631 50471 SimplyLoad® Ladders (Cat. No.) 50331 50326 50327 50328 50333 50329 50336 50337 NA NA NA Number of 25 50 10 30 21 16 5 5 9 13 9 Fragments Size Range 20 bp – 20 bp – 100 bp – 10 bp – 100 bp – 500 bp – 100 bp – 100 bp – 50 bp – 50 bp – 1 kb – 500 bp 1,000 bp 1,000 bp 3,000 bp 12 kb 8 kb 1,000 bp 1,000 bp 1,000 bp 2,500 bp 10 kb 500 bp 1,000 bp 1,000 bp 3,000 bp 12 kb 8,000 bp 1,000 bp 1,000 bp 1,000 bp 2.5 kb 10 kb 480 bp 980 bp 900 bp 2,900 bp 11 kb 7,500 bp 700 bp 700 bp 700 bp 2 kb 7 kb 460 bp 960 bp 800 bp 2,800 bp 10 kb 7,000 bp 500 bp 500 bp 525 bp 1.5 kb 5 kb 440 bp 940 bp 700 bp 2,700 bp 9 kb 6,500 bp 200 bp 200 bp 500 bp 1250 bp 4 kb 420 bp 920 bp 600 bp 2,600 bp 8 kb 6,000 bp 100 bp 100 bp 400 bp 1 kb 3 kb 400 bp 900 bp 500 bp 2,500 bp 7 kb 5,500 bp 300 bp 700 bp 2.5 kb 380 bp 880 bp 400 bp 2,400