International Scholarships and Financial Aid

Wednesday, 27 June 2007

4th annual RNAi Europe conference

RNAi Europe, 20-21 September, Barcelona, Spain

 4th annual RNAi Europe conference and exhibition,  will be held in Barcelona, Spain. The agenda will include an excellent   list of influential speakers, and we are expecting 30+ exhibitors.   A tutorial to review RNAi and MicroRNA: Market Landscape and Emerging Opportunities will be held on the 21 September.
http://www.selectbiosciences.com/conferences/rnaieurope07/index.aspx

Sunday, 24 June 2007

RT PCR Protocols and Analysis

Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample

Maria Marone1*, Simona Mozzetti1, Daniela De Ritis1, Luca Pierelli1 and Giovanni Scambia1

1 Department of Gynecology and Department of Hematology. Catholic University, L.go A. Gemelli 8, 00168 Rome. Italy.
* To whom correspondence should be addressed: Maria Marone, Department of Gynecology and Department of Hematology. Catholic University, L.go A. Gemelli 8, 00168 Rome. Italy. Email: maria.marone@tiscalinet.it

Link

http://www.biologicalprocedures.com/bpo/arts/1/20/m20.htm

RT-PCR Protocol

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Other PCR Procedures

from

Jack Vanden Heuvel
Penn State University
Department of Veterinary Science
and Molecular Toxicology Program

Link

RT-PCR Protocol

Protocol for competitive RT-PCR

This protocols is from the lab of Dieter Kaufmann, Department of Human Genetics, University of Ulm, Germany

Link

RT-PCR Protocols ebook

RT-PCR Protocols (Methods in Molecular Biology)

This RT-PCR protocol book describes the detail of novel, useful, and
interesting RT-PCR applications.

protocols available for

highly sensitive detection and quantification of gene expression, the in situ localization of gene,
expression in tissue, and the cloning of genes,

as well as for analyzing T-cell clones and the differential expression of genes,

including laser-capture microdissection (LCM),

real-time and quantitative PCR,

microarray technology, cDNA cloning.

Book Info:
Published in 2002
Published by Humana press
Author Joseph OConnell
ISBN 0896038750  Size 4.30MB

Link

WordWeb-English thesaurus and dictionary for Windows

WordWeb is a English thesaurus and dictionary for Windows. New Version 5 - now with fast one-click look up, web-reference tabs, bookmarks, Vista support. Highly useful for non-native English speakers, GRE, TOEFL and other examinations.

Link

Thomson ISI ResearchSoft EndNote XI v11.0

Thomson ISI ResearchSoft EndNote XI v11.0 is world's most popular bibliographic software.

Link

Clinician's Guide To Medical Writing ebook

  Link

Saturday, 23 June 2007

Handbook of Cardiac Electrophysiology: A Practical Guide to Invasive EP Studies and Catheter Ablation ebook

By Francis D. Murgatroyd, Andrew D. Krahn, George J. Klein, Raymond K. Yee, 

  • Publisher:   Remedica Publishing
  • Number Of Pages:   250
  • Publication Date:   2002-02
  • Sales Rank:   184297
  • ISBN / ASIN:   1901346374
  • EAN:   9781901346374
  • Binding:   Hardcover
  • Manufacturer:   Remedica Publishing
  • Studio:   Remedica Publishing
  • Average Rating:   4.5
  • Total Reviews:   2

Link

Computational Cardiology: Modeling of Anatomy, Electrophysiology, and Mechanics ebook

Computational Cardiology: Modeling of Anatomy, Electrophysiology, and Mechanics (Lecture Notes in Computer Science)
By Frank B. Sachse
Publisher: Springer
Number Of Pages: 17
Publication Date: 2004-05-27
Sales Rank: 933074
ISBN / ASIN: 3540219072
EAN: 9783540219071
Binding: Paperback
Manufacturer: Springer
Studio: Springer

Link

Analytical Biochemistry (3rd Edition) ebook

Paperback: 504 pages

  • Publisher: Prentice Hall; 3 edition (December 31, 1997)
  • Language: English
  • ISBN-10: 058229438X
  • Now an established handbook of principles and techniques, this text develops an understanding of the relevance of four fundamental properties of the analyte: shape, polarity, charge and size, to the three key types of analysis: separation, identification and quantification.

    Link

    Reviews of Physiology, Biochemistry and Pharmacology ebook

    Publisher: Springer
    Number Of Pages: 112
    Publication Date: 2005-04-06
    Sales Rank:
    ISBN / ASIN: 3540240128
    EAN: 9783540240129
    Binding: Hardcover
    Manufacturer: Springer
    Studio: Springer

    Link

    Nutritional Biochemistry of the Vitamins eBook

    Nutritional Biochemistry of the Vitamins

  • Publisher: Cambridge University Press
  • Number Of Pages: 512
  • Publication Date: 2003-09-15
  • Sales Rank: 1193213
  • ISBN / ASIN: 0521803888
  • EAN: 9780521803885
  • Binding: Hardcover
  • Manufacturer: Cambridge University Press
  • Studio: Cambridge University Press
  • Link

    Clinical Studies in Medical Biochemistry E book

    Clinical Studies in Medical Biochemistry
  • Publisher: Oxford University Press, usa
  • Number Of Pages: 392
  • Publication Date: 2006-08-07
  • Sales Rank: 587316
  • ISBN / ASIN: 019517688X
  • EAN: 9780195176889
  • Binding: Paperback
  • Manufacturer: Oxford University Press, USA
  • Studio: Oxford University Press, USA
  • Book Description:
  • Link

    Saturday, 16 June 2007

    Drug Discovery & Development of Innovative Therapeutics (DDT) World Congress to take place on August 6-9, 2007 in Boston, MA.

    Drug Discovery & Development of Innovative Therapeutics (DDT) World Congress to take place on August 6-9, 2007 in Boston, MA.

    This year DDT returns with a new name, new focus and new content. DDT 2007 focuses on scientific innovations, new approaches and business strategies that are helping companies to accelerate movement of compounds into the clinic. This year the World Congress theme is “Building Your Drug Pipeline, Faster, Better and Cheaper from Discovery to Clinical Proof-of-Concept” which reflects the ultimate goal of the pharmaceutical and biotechnology industries to increase R&D efficiency and bring innovate medicines to the marketplace as quickly as possible.

    http://www.drugdisc.com/default.asp?page=register&source=D3201SE1

    ENCODE-Encyclopedia Of DNA Elements

    ENCODE= the Encyclopedia Of DNA Elements

    ENCODE was started in September 2003 to identify all functional elements in the human genome sequence by the National Human Genome Research Institute (NHGRI).

    Project has three phases.

    1. a pilot project phase-testing and comparing existing methods to rigorously analyze a defined portion of the human genome sequence

    2. a technology development
    3. a planned production phase

    Full details can be found at http://www.genome.gov/10005107

    This week Nature published the results of the pilot phase of ENCODE project.


    Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project
    The ENCODE Project Consortium*
    http://www.nature.com/nature/journal/v447/n7146/pdf/nature05874.pdf

    One of the main conclusions they are pointing to is that there is a lot of places wherethere appears to be conservation of function (transcription and it's regulation) without conservation of sequence. Very interesting.. but long way to go.

    wellcome trust scientific conferences

    wellcome trust scientific conferences

    Scientific conferences (up to 300 delegates) are held at the Wellcome Trust Conference Centre. These are mainly open, registration-fee paying conferences run on a not-for-profit basis. These conferences may be fully administrated by the Trust, or co-organised and sponsored with other bodies such as Cold Spring Harbor Laboratory and the European Science Foundation.
    Should you wish to discuss with the Trust a potential topic for a scientific conference, please contact Dr Debbie Carley – d.carley@wtconference.org.uk – in the first instance.

    http://www.wellcome.ac.uk/node6233.html

    Current Protocols in Molecular Biology

    Current Protocols in Molecular Biology

    Current Protocols in Molecular Biology
    ISBN: 047150338X
    Author: Roger Brent / Robert E. Kingston / J. G. Seidman / Kevin Struhl / Frederick M. Ausubel / Virginia Benson Chanda / David D. Moore / J.G. Seidman / F.M. Ausubel
    Publisher: John Wiley & Sons Inc
    Edition: ringbou edition (December 4, 2003)
    ISBN: 047150338X
    Ring-bound: 1600 pages
    URL: /http://www.amazon.com/exec/obidos/redirect?tag=songstech-20&path=ASIN%2F047150338X
    Summary:

    Current Protocols in Molecular Biology, the first Current Protocols publication, remains the international standard by which all other lab manuals are judged. Basic methods for DNA preparation and isolation, library screening, and sequencing have been joined by more advanced procedures detailing DNA-protein interactions, yeast manipulation, and phosphorylation analyses. From basics to the cutting edge, CPMB is the only resource you need for successful experiments.

    Link

    Protocol for Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation

    DNA Plasmid is isolated from 1-2 ml bacterial cultures by alkali and SDS treatment.

    Link

    Friday, 15 June 2007

    Bioinformatics: Sequence and Genome Analysis-E Book

    • Paperback: 692 pages
    • Publisher: Cold Spring Harbor Laboratory Press; 2nd edition (July 2004)
    • Language: English

    As more species' genomes are sequenced, computational analysis of these data has become increasingly important. The second, entirely updated edition of this widely praised textbook provides a comprehensive and critical examination of the computational methods needed for analyzing DNA, RNA, and protein data, as well as genomes. The book has been rewritten to make it more accessible to a wider audience, including advanced undergraduate and graduate students. New features include chapter guides and explanatory information panels and glossary terms. New chapters in this second edition cover statistical analysis of sequence alignments, computer programming for bioinformatics, and data management and mining. Practically oriented problems at the ends of chapters enhance the value of the book as a teaching resource. The book also serves as an essential reference for professionals in molecular biology, pharmaceutical, and genome laboratories.

    Link

    The $800 Million Pill-Ebook

    Link

    SV40 Protocols-Ebook (Methods in Molecular Biology)

    SV40 Protocols (Methods in Molecular Biology) -
    Book Properties
    ISBN: 0896036537
    Title: SV40 Protocols (Methods in Molecular Biology)
    Author:
    Publisher: Humana Press

    Link

    MicroRNA Protocols (Methods in Molecular Biology)

    MicroRNA Protocols (Methods in Molecular Biology)

  • Publisher: Humana Press
  • Number Of Pages: 272
  • Publication Date: 2006-04-01
  • Sales Rank: 1248190
  • ISBN / ASIN: 1588295818
  • EAN: 9781588295811
  • Binding: Hardcover
  • Manufacturer: Humana Press
  • Studio: Humana Press
  • Book Description:

    MicroRNA Protocols provides diverse, novel, and useful descriptions of miRNAs in several species, including plants, worms, flies, fish, chicks, mice, and humans. These include some useful adaptations and applications that could be relevant to the wider research community who are already familiar with the identification of miRNAs. This volume will stimulate the reader to explore diverse ways to understanding the mechanism in which miRNAs facilitate the molecular aspects of the biomedical research.

  • Link

    pass:lekar

    Thursday, 14 June 2007

    Protocol of Hemacytometer Use

    The following protocol is written as an aide in the use of a hemacytometer for determining the concentration of
    cells in a suspension.

    Download

    Protocols Ebook for Cryopreservation and Freeze-Drying (Methods in Molecular Biology)

    Cryopreservation and Freeze-Drying Protocols (Methods in Molecular Biology)

    • Publisher: Humana Press
    • Number Of Pages: 254
    • Publication Date: 1995-03-24
    • Sales Rank: 2247135
    • ISBN / ASIN: 0896032965
    • EAN: 9780896032965
    • Binding: Paperback
    • Manufacturer: Humana Press
    • Studio: Humana Press

    • Book Description:

      This book provides detailed protocols for all the latest methodologies used to assure the long-term biostorage of a diverse range of biological materials.
      Developed in expert laboratories, the protocols have been painstakingly perfected over the years to provide time-tested, step-by-step instructions that ensure robust and reproducible results. Each protocol deals with the preservation of an organelle, cell, or tissue type, and is accessible even to the nonspecialist because of its cookbook approach. Novel protocols can be readily developed with the help of the "hands-on" Notes sections.
      Cryopreservation and Freeze-Drying Protocols is an indispensable reference work for both the individual researcher, and all those who want to establish or improve biostorage systems in their laboratories. Its applications range from microbial culture collections, botanic gardens, and zoos to animal husbandry, aquaculture, medicine, human fertilization, and cell and molecular biology.

    Link

    PCR Protocols-Methods in Molecular Biology EBook

    PCR Protocols: Current Methods and Applications (Methods in Molecular Biology)

  • Publisher: Humana Press
  • Number Of Pages: 392
  • Publication Date: 1993-01
  • Sales Rank: 914567
  • ISBN / ASIN: 0896032442
  • EAN: 9780896032446
  • Binding: Spiral-bound
  • Manufacturer: Humana Press
  • Studio: Humana Press

    Link

  • Pass: econiches

    Biology E Book, Sixth Edition, Neil A. Campbell, Jane B. Reece

    Product Details
    Book Publisher: Benjamin Cummings (11 December, 2001)
    ISBN: 0805366245
    Book author: Neil A. Campbell, Jane B. Reece
    Amazon Rating: 4.5
    Book Description:
    The Sixth Edition of BIOLOGY by Neil Campbell and Jane Reece builds upon the earlier versions' dual goals to both help readers develop a conceptual appreciation of life within the context of integrating themes, and to inspire readers to develop more positive and realistic impressions of science as a human activity.
    The authors have thoroughly updated each of the book's eight units to reflect the existing progress in our understanding of life at its many levels, from molecules to ecosystems. Examples of updated content include the Human Genome Project, the revolution in systematics, HIV as a research model in evolutionary biology, the role of cell-signaling pathways in plant responses, new frontiers in neurobiology, and experimental approaches that are advancing ecology. To assure accurate representation of each field of biology, a team of stellar specialists has worked with the authors in updating every unit.
    An innovative design breakthrough ensures that the art is as current as the content. Guided Tour diagrams explicitly guide readers through the more challenging figures, succinctly explaining key structures, functions, and steps of processes within the figure, reducing the need to look back and forth between legend and art. It's as if an instructor were looking over the reader's shoulder and clarifying each part of a figure! Guided Tour commentary is set in blue, making it easy to differentiate these explanations from ordinary labels and keeping the figure itself clear and uncluttered. For college instructors and students.

    http://mihd.net/198oy7

    password: lekar

    The Open Laboratory-The Best Writing on Science Blogs 2006

    Blogmania has been global and its roots can be found deep in to the hardcore science. This week's Nature article "Blogger UnitThe Open Laboratory: The Best Writing on Science Blogs 2006 by Bora Zivkovic, Editor (Book) in Medicine & Sciencestarted another wave of science blogging by describing about the book "The Open Laboratory: The Best Writing on Science Blogs 2006". The Book is written by the a US biologist-blogger Bora Zivkovic. He is one of the highly successful science blogger (Bora's blog*) and actively engaged in promoting science blogging.

    The book teach us about the science blogging anthology. Having look at the book preview, I personally find it interesting but don't think its good for new science bloggers. Because blogging is not about scientific writing and publishing research.

    People love write a blog because they enjoy personal freedom which we don't have in scientific world...

    Anyways enjoy the book...

    Wednesday, 13 June 2007

    How MRI works? Ebook: An Introduction to the Physics and Function of Magnetic Resonance Imaging

    How does MRI work?: An Introduction to the Physics and Function of Magnetic Resonance Imaging

    This thoroughly revised second edition succinctly introduces the physics and function of magnetic resonance imaging. All important and clinically relevant aspects are presented in a clearly structured manner. The emphasis is on practical information including the latest trends and developments that are relevant for MRI in the clinical setting. The opening chapters describe the underlying physical principles of the MR experiment and the basic pulse sequences commonly used in clinical MRI. Other chapters are dedicated to more advanced techniques such as parallel imaging and cardiovascular MR imaging. The book is rounded out by chapters on MR contrast media, artifacts, high-field imaging, and safety concerns. An extensive glossary offers rapid access to the most important MRI terminology. The book is intended for readers looking for an easy to understand and concise introduction to this fascinating yet somewhat complex imaging modality at the beginning of their MRI training.

    Link

    Standard PCR Protocol

    Standard PCR Protocols

    This protocol is link to Molecular Biology Techniques Manual
    Third Edition
    Edited by:
    Vernon E Coyne, M Diane James, Sharon J Reid and Edward P Rybicki

    Read Full Protocol

    Overlap Extension PCR

    Overlap Extension PCR is used to create long DNA fragments from short ones.

    or used for Engineering the replication of target DNA through cloning, or changing its genetic code through mutations

    PCR amplify the necessary fragments, using polymerase enzyme. They should have about 15-25 bp overlaps. Use oligo Tm calculators to figure out their annealing temp.
    Clean up or gel extract the correct size band. Use cleaned up fragments as "template". Unlike normal PCR, about 1/2 to 3/4 volume of the extension reaction should be template.
    Use proofreading enzyme for extension. Run 3 reactions of 10,15 and 30 PCR cycles without end primers. (Template extension step) Add end primers, then continue cycling for another 15-20 rounds. Gel extract the correct fragment. Clone into a your desired vector.

    check out the latest Nature Methods Protocol
    http://www.nature.com/nmeth/journal/v4/n5/pdf/nmeth0507-455.pdf

    Primer Sets and PCR Manual


    This 24-page brochure details each step of the Polymerase Chain Reaction process with technical information for basic PCR techniques, methods, applications and polymerase choices. You will want to keep this new booklet close at hand because it also includes FAQs, references and a troubleshooting guide.


    Oxford Handbook of Clinical and Laboratory Investigation-Ebook

    Oxford Handbook of Clinical and Laboratory Investigation (Oxford Handbooks Series)
    By
    Publisher: Oxford University Press, USA
    Number Of Pages: 838
    Publication Date: 2005-11-18
    Sales Rank: 847502
    ISBN / ASIN: 0198566638
    EAN: 9780198566632
    Binding: Paperback
    Manufacturer: Oxford University Press, USA
    Studio: Oxford University Press, USA
    Average Rating: 5
    Total Reviews: 1
    Book Description:
    Modern medicine is highly complex and investigations are a key part of the diagnostic process. With major advances in technology there are thousands of clinical and laboratory tests available. This book provides a patient-oriented approach to investigation (first part of book) where key symptoms and signs are described along with tests that may be of value in reaching a diagnosis. The remainder of the book is specialty-centred and provides a comprehensive review of all available tests within a given subject. The book emphasises which tests are of value, when tests are not likely to be helpful, along with pitfalls in the interpretation of results. The aim is of the book is to provide a more rational method of investigation and prevent over-investigation which is expensive for the hospital and unpleasant for the patient. The contributors are all active clinicians who are engaged in medical practice, so appreciate the problems faced by junior doctors. The book should also be of value to senior medical students who will be facing finals examinations, and who will soon be on the wards and responsible for ordering tests on their patients.

    Link

    Oxford textbook of surgery Ebook

    Second author, William C. Wood, is with Emory Univ., Atlanta. Comprehensive reference for general and specialist surgeons and residents. Includes halftone and color illustrations and extensive references. Majority of contributors are from either Oxford Univ., UK or Harvard Medical School, Boston, MA. Previous edition:
    c1994.

    Link1

    Link2

    Link3

    Link4

    pass:rafcm

    Primary Care Pediatrics-Ebook

    Primary Care PediatricsThis pediatric primary care text takes a family centered approach, examining issues from the perspective of the child and the caregiver. Unlike most primary care texts, it blends traditional health care with complementary therapies. Throughout, it identifies needs based on culture and ethnicity and lists community resources-including websites--where families can obtain more information or help. Features include: Case Studies, Clinical Pearls, and Clinical Warnings.

     

    Link

    Pediatrics 2007-Ebook by Paul D., M.D. Chan

    Title: Pediatrics 2007
    Author: Paul D., M.D. Chan
    Publisher: Current Clinical Strategies
    Publication Date: 2006-08-08
    Number Of Pages: 128

    Link

    Pass: MeDiCiNe_DoCtOrS

    Obstetrics and Gynaecology E-Book (Lecture Notes)

    Obstetrics and Gynaecology (Lecture Notes)

    Lecture Notes: Obstetrics and Gynaecology provides a concise introduction to obstetrics and gynaecology for medical students and junior doctors. Six sections examine the female development from the early years to old age. The text starts with a section on Basic Science. Self-assessment questions are found throughout the text to enhance knowledge and understanding.Part 1 looks at female reproductive anatomy and physiology in detail. Part 2 covers the puberty and menstrual problems of young women, sub fertility, pregnancy prevention and introduces benign diseases, genital tract infections and sexual problems.Part 3 examines the reproductive years. Detailed coverage of the mother and foetus in pregnancy includes problems and diseases in pregnancy, labour and the newborn child development. Part 4 covers the mature woman, and includes abnormal vaginal blood loss, pelvic pain, breast disease and screening for gynaecological cancer.Part 5 discusses the older woman and malignant gynaecological conditions, the menopause and pelvic floor disorders. Part 6 provides an audit of Obstetrics and Gynaecology, looking at statistics in reproductive medicine and gives the answers to self-assessment questions found throughout the text.Lecture Notes: Obstetrics and Gynaecology is written specifically for medical students, junior doctors on foundation programmes, midwives and General Practitioners.Review quotes on Previous Editions"Well edited, concise, and a useful source of information."2nd Opinion - Edinburgh Student Gazette"Thankfully these are nothing like my lecture notes. This book is packed with useful info."Surgo, Glasgow University Medical Journal"A really easy book to read, useful in enabling students to grasp the basics."Northwing - Sheffield Medical Magazine
    Lecture Notes: Obstetrics and Gynaecology provides a concise introduction to obstetrics and gynaecology for medical students and junior doctors. Six sections examine the female development from the early years to old age. The text starts with a section on Basic Science. Self-assessment questions are found throughout the text to enhance knowledge and understanding.Part 1 looks at female reproductive anatomy and physiology in detail. Part 2 covers the puberty and menstrual problems of young women, sub fertility, pregnancy prevention and introduces benign diseases, genital tract infections and sexual problems.Part 3examines the reproductive years. Detailed coverage of the mother and foetus in pregnancy includes problems and diseases in pregnancy, labour and the newborn child development. Part 4covers the mature woman, and includes abnormal vaginal blood loss, pelvic pain, breast disease and screening for gynaecological cancer.Part 5 discusses the older woman and malignant gynaecological conditions, the menopause and pelvic floor disorders. Part 6 provides an audit of Obstetrics and Gynaecology, looking at statistics in reproductive medicine and gives the answers to self-assessment questions found throughout the text.Lecture Notes: Obstetrics and Gynaecology is written specifically for medical students, junior doctors on foundation programmes, midwives and General Practitioners. Review quotes on Previous Editions "Well edited, concise, and a useful source of information."2nd Opinion - Edinburgh Student Gazette"Thankfully these are nothing like my lecture notes. This book is packed with useful info." Surgo, Glasgow University Medical Journal "A really easy book to read, useful in enabling students to grasp the basics."Northwing - Sheffield Medical Magazine

    Link

    Interventional Ultrasound (Ebook) in Obstetrics, Gynaecology and the Breast

    Interventional Ultrasound in Obstetrics, Gynaecology and the Breast

    Author: Joaquin Santolaya-Forgas; Didier L�mery
    Publisher: Blackwell Science
    Publication Date: 1998-11-15
    Number Of Pages: 276
    Average Amazon Rating:
    Book Id 38394

    Link

    Pass: Lekar

    General Practice EBook of Murtagh, 3rd edition

    General Practice

    General Practice is one of McGraw-Hill Australia's best selling, most reputable and well-established titles. It is the 'Bible' for general practitioners and medical students of community medicine, written by Australia's most respected GP, John Murtagh.

    Link

    Pass: spiderman

    Oxford Handbook of General Practice-Ebook

    A lifeline for the busy GP, the Oxford Handbook of General Practice covers the whole of general practice. It includes hands-on advice and allows rapid access to information to help with any day-to-day problems which might arise in general practice. The general practice section has been revised to include the new General Medical Services contract and appraisal and revalidation. There are new chapters on complementary medicine, chronic disease management and elderly care and increased emphasis on evidence based medicine, sports medicine and practice in a multicultural society. Pointers to further information for GPs and advice and support for patients, which can be easily accessed from the GP surgery, are included throughout. The layout of the handbook has been redesigned with the use of colour-coding to aid quick reference and additional flow charts, tables and diagrams have been included to make information easier to access.

    Link

    Tuesday, 12 June 2007

    Back Translation of Protein-Online Software

    Backtranslation from Entelechon Server translates a protein sequence back to a nucleotide sequence .

    Translate Now

    how to translate all 6 frames of a nucleotide sequence to protein sequences?

    Use online traslation tool from Expasy Server. This a tool allows the translation of a nucleotide (DNA/RNA) sequence to a protein sequence in all 6 frames.

    Translate Now

    Basic Immunology Ebook: Functions and Disorders of the Immune System Code- by Abul K. Abbas, Andrew H. Lichtman

    The 2nd of this popular text emphasizes the fundamental concepts and principles of human immunology that students need to know, without overwhelming them with extraneous material. It leads the reader to a firm understanding of basic principles, using full-color illustrations; short, easy-to-read chapters; color tables that summarize key information clinical cases; and much more-all in a conveniently sized volume that's easy to carry. The New Edition has been thoroughly updated to reflect the many advances that are expanding our understanding of the field.

    Link

    Encyclopedia of Immunology-Ebook, Second Edition

    With more than 700 expert authors from 22 different countries, the Encyclopedia of Immunology, Second Edition is the largest comprehensive reference source of current immunological knowledge available. It provides a broad scope and high level of expertise to the many aspects of the field of immunology and related areas, including microbiology, virology, and parasitology. Arranged into 31 subject areas with extensive cross-referencing and subject indexes in each volume, the Encyclopedia is easy-to-use for virtually any researcher, regardless of his or her field. Concise definitions of the subject area also introduce each entry. The Second Edition includes timely and thorough updates for all articles from the First Edition, more than 60 new entries, a glossary of immunological terms in each volume, a total of 500 figures and tables, and new color plates sections.

    Key Features
    * Four volumes, each containing a subject index
    * Approximately 630 different articles
    * More than 700 expert contributors from 22 different countries
    * Coverage of 31 different subject areas
    * Concise definitions of the subject to introduce each entry
    * Further reading lists at the end of each entry
    * Extensive cross-referencing
    * Entries arranged in a single A-Z list for easy access
    * Easy-to-read double-column format
    * More than 500 figures to complement the text
    * More than 60 new articles
    * A glossary of immunological terms in each volume
    * A color plate section in each volume

    Link1

    Link2

    Link3

    Immunology Guidebook-E Book

    The Immunology Guidebook provides an easily accessible text-reference to the more
    up-to-date and difficult concepts in the complex science of immunology. It aims to demystify basic concepts and specialised molecular and cellular interactions. Its 18 chapters offer a logical and sequential presentation where much of the data is displayed in carefully designed tables. This book is intended for immunology students, researchers, practitioners and basic biomedical scientists.
    * Tables provide a quick reference to difficult to find immunology data
    * A distillate of the latest information on immunogenetics of the human MHC associated with tissue transplantation
    * Information boxes featurw related web resources

    Link

    Introduction to Bioinformatics-Ebook by Teresa Attwood

    Introduction to BioinformaticsTeresa Attwood (Author), David Parry-Smith (Author)

     

    Teresa Attwood is a well known professor in Bioinformatics at University of Menchester. She has been also published in Science.

    Download

    DNA Microarray Technology. What is it and how is it useful?

    This is very simple easy to understand manual of
    DNA Microarrays written by MIT Laboratory Protocols. Highly useful for
    the teachers.


    http://mit.edu/biology/www/outreach/precollege/DNAmicroarray.pdf

    Isolation of Acetylcholinesterase and lectin Protocols Links*

    Identificationandisolationoflectinnu.pdf
    238k

    IsolationandCharacterizationofRanaca.pdf
    1M

    Isolationandpartialcharacterizationo.pdf
    896k

    Isolationandpropertiesofalectinfromt.pdf
    287k

    Isolationofalectinfromthepericarpofp.pdf
    521k

    proteinpurificationmicrotomacro.pdf
    1M

    PurificationandCharacterizationofanN.pdf

     

    *Please note that these links are from other websites and we do not host these files on our server.  Links will be deleted if reported abuse. please email us at laboratory.helpdesk@gmail.com

    Monday, 11 June 2007

    Oncogenomics-E Book: Molecular Approaches to Cancer

    Oncogenomics: Molecular Approaches to Cancer

    Cancer is not a single genetic disease but, rather, hundreds of diseases consisting of various combinations of genetic alterations. Many types of genetic alterations contribute to neoplastic transformation. The evidence for this statement has now become common knowledge. In a similar vein, almost everybody believes that genome sequencing is paving the way to a revolution in biology and medicine in general but in oncology in particular. We all mention imatinib and a few monoclonal antibodies when we want to demonstrate that targeted therapies are already more than just a vision. We are, however, still unable to foresee clearly the role

    genomics will play in the prevention, diagnosis, and treatment of cancer in the next 10 or 15 years. This uncertainty stems mainly from a dearth of clinical data, and most of the data we do have are retrospective and therefore most probably biased. As stated in its introduction, this book is probably the first of its kind devoted to the genomics of cancer. I approached it with mixed feelings: on the one hand, I was hoping to find answers to some of my questions, mainly because the span of the book reaches from molecular profiling to model systems for discovering and validating drug targets, and from molecularly targeted pharmacology to clinomics. The latter is a term coined by Daniel Von Hoff et al., whose chapter in this book defines clinomics as the application of genomics to patient care. On the other hand, I approached the book with some
    skepticism, since the field is moving so rapidly that I was afraid to find once again a book that was already too old or just provided reviews that everyone in the field has already read. But I was pleasantly surprised to find a book that is extremely rich in information and detail but is not

    more specialized than necessary. One might criticize certain choices the editors have made -- for instance, providing a discussion of the role of proteomics and genomics in bladder cancer but not in colorectal and lung cancer. This, however, is not very important, since the book was not written for physicians who seek an overview of the latest discoveries in the molecular biology of a specific cancer type. Oncogenomics is very good reading for oncologists who would like to understand what is happening in the laboratory and in the preclinical setting, especially in terms of techniques and research approaches. The book is also worth reading for basic and clinical scientists who intend to plan translational and clinical studies that entail oncogenomics. The essence of the book is to bring together scientists from various specialties and share the same

    language, a prerequisite for the next "leap forward." It seems logical that the book concludes with an essay by A.C. von Eschenbach, the director of the National Cancer Institute, who gives his vision of oncology in 2015. Franco Cavalli, M.D., F.R.C.P.

    Download

    Pass: dmc@lekar

    TIGR-GlimmerM- a Gene Structure Prediction Tool-Download

    TIGR-GlimmerM: A gene finder derived from Glimmer, but developed specifically for eukaryotes. It is based on a dynamic programing algorithm that considers all combinations of possible exons for inclusion in a gene model and chooses the best of these combinations.
    Download Glimmer Via FTP

    TIGR-Glimmer-a Gene Structure Prediction Tool

    TIGR-Glimmer: Glimmer are for identification of possible genes for prokaryotes sequences . It is based on interpolated Markov models to identify the coding regions and distinguish them from noncoding DNA.
    Download Glimmer Via FTP

    TIGR-AAT package- Download

    TIGR-AAT package(A tool for analyzing and annotating genomic sequences): The AAT package includes two sets of programs, one set (DPS/NAP) for comparing the query sequence with a protein database, and the other (DDS/GAP2) for comparing the query with a cDNA database (Huang et al., 1997).
    Download AAT package via FTP

    IGR-MUMmer

    IGR-MUMmer(fast alignment of large-scale DNA and protein sequences):A system for aligning whole genome sequences. Using an efficient data structure called a suffix tree, the system is able rapidly to align sequences containing millions of nucleotides.
    Download MUMer Via FTP

    Clustal X Download

    Clustal X: Clustal X is a new windows interface for the ClustalW multiple sequence alignment program. It provides an integrated environment for performing multiple sequence and profile alignments and analysing the results. The sequence alignment is displayed in a window on the screen. A versatile coloring scheme has been incorporated allowing you to highlight conserved features in the alignment. The pull-down menus at the top of the window allow you to select all the options required for traditional multiple sequence and profile alignment.
    Download Clustax (Windows OS)

    EBI-Clustal W Download

    EBI-Clustal W is a general purpose multiple sequence alignment program for DNA or proteins.It produces biologically meaningful multiple sequence alignments of divergent sequences. It calculates the best match for the selected sequences, and lines them up so that the identities, similarities and differences can be seen.
    Download EBI-Clustal W via FTP

    EBI- Fasta Download

    Provides sequence similarity searching against nucleotide and protein databases using the Fasta programs. Fasta can be very specific when identifying long regions of low similarity especially for highly diverged sequences.
    Download EBI-Fasta executable via FTP

    Sigma Plot 10.0 (Stats-Scientific Graphing Software)

     

    SigmaPlot software helps you quickly create exact graphs
    SigmaPlot graphing software from SYSTAT takes you beyond simple spreadsheets to help you show off your work clearly and precisely. With SigmaPlot, you can produce high-quality graphs without spending hours in front of a computer. SigmaPlot offers seamless M'zoft Office integration, so you can easily access data from M'zoft Excel spreadsheets and present your results in M'zoft PowerPoint? presentations.

    I'll say Highly Powerful Software

    http://www.systat.com/products/sigmaplot/

    part 1 link

    part 2 link

    Sunday, 10 June 2007

    Microbiology by Prescott-EBook

    Heyy guys.. check out the link of Microbiology book

     

    Download Part1

     

    Download Part2

     

    Download Part3

    Friday, 8 June 2007

    Microarrays Protocols

    Gernal Consideration Microarrays

    Producing fluorescence labeled cDNA

    Washing the hybrid Miroarrays Protocol

    SDS Page-Mini-PROTEAN 3 Cell Instruction Manual

    I personally find this BioRad manual very useful in SDS page. I even use this protocol to run Hoofer cabinets..

    Download

    High-Yield Biostatistics (High-Yield Series)-E-Book

    Synopsis
    Part of the successful High-Yield "TM" Series, High-Yield "TM" Biostatistics, Second Edition explains concepts, provides examples, and covers the complete range of biostatistics material that can be expected to appear on the USMLE Step 1. New to this edition are references to evidence-based medicine, and information updated to reflect changes in the current USMLE examinations.
    Table of Contents
    Descriptive statistics - Inferential statistics - Hypothesis testing - Correlational techniques - Research methods - Statistics in epidemiology - Statistics in medical decision making - Ultra-high-yield review

    Download

    Primer of Biostatistics-E-Book

    Primer of Biostatistics

    Extremely popular, this student-friendly text presents the practical areas of statistics in terms of their relevance to medicine and the life sciences. Includes many illustrative examples and challenging problems that reinforce the author's unique and intuitive approach to the subject. The new edition features a new two-color design, examples taken from current biomedical literature, and review questions within each chapter.

    Download Part1

    Download Part2

    Download Part3

    Handbook of Statistics in Clinical Oncology-E-Book

    Handbook of Statistics in Clinical Oncology

    Chapman & Hall/CRC
    ISBN: 0824723392
    640pages
    2006-
    Pdf-English
    size:17,42 Mb

    Book Description
    A compendium of cutting-edge statistical approaches to solving problems in clinical oncology, this book focuses on clinical trials in phases I, II, and III, proteomic and genomic studies, complementary outcomes and exploratory methods. Cancer Forum called the first edition a "good reference book for statisticians who will be designing and analyzing cancer trials." The second edition includes over 1000 references, more than forty world-renowned contributors, and 300 equations, tables, and drawings. Completely revised while keeping the features that made the first edition a bestseller, this is the best single source for up-to-date statistical approaches to research in clinical medicine.
    Features:
    Provides a comprehensive discussion of sample size
    Explores analytical problems generated by controlling treatment costs and maintaining quality of life
    Demonstrates the breadth and depth of current activity in the field of survival analysis
    Includes recommendations and pointers for free software that allows you to implement programs
    Sets the limits on what can and cannot be concluded from single and multiple clinical trials
    Contents
    Phase I Trials
    Phase II Trials
    Phase III Trials
    Exploratory Analysis and Prognostic Factors
    High-Throughput Data and Bioinformatics
    Interpreting Clinical Trials

    Download

    Handbook of Statistics 18: Bioenvironmental and Public Health Statistics-E-Book

    Handbook of Statistics 18: Bioenvironmental and Public Health Statistics

    Handbook of Statistics 18: Bioenvironmental and Public Health Statistics (Techniques and Instrumentation in Analytical Chemistry)
    Product Details
    �Book Publisher: Elsevier Publishing Company (01 April, 2000)
    �ISBN: 0444829008
    �Book author: Pranab Kumar Sen
    Book Description:
    Hardbound. In this volume of the Handbook of Statistics with the primary focus on bioenvironmental and public health statistics, a rather off-beat approach has been taken, wherein biostatistical methods that are relevant to the dissemination of bioenvironmental and public health investigations have been thoroughly emphasised, and placed side by side with the fruitful applications. One aspect of statistical methodology that merits special appraisal is the extent of appropriateness of some standard statistical tools in such non-standard applications, and much of the deliberation in this volume is geared to alternative non-standard and application oriented methodology that have been developed to suit better bioenvironmental and public health studies.

    Download

    Statistical Methods in Bioinformatics: An Introduction-E-Book

    • Hardcover: 588 pages
    • Publisher: Springer; 2 edition (September 30, 2005)
    • Language: English
    • ISBN-10: 0387400826

    Advances in computers and biotechnology have had a profound impact on biomedical research, and as a result complex data sets can now be generated to address extremely complex biological questions. Correspondingly, advances in the statistical methods necessary to analyze such data are following closely behind the advances in data generation methods. The statistical methods required by bioinformatics present many new and difficult problems for the research community.

    This book provides an introduction to some of these new methods. The main biological topics treated include sequence analysis, BLAST, microarray analysis, gene finding, and the analysis of evolutionary processes. The main statistical techniques covered include hypothesis testing and estimation, Poisson processes, Markov models and Hidden Markov models, and multiple testing methods.

    The second edition features new chapters on microarray analysis and on statistical inference, including a discussion of ANOVA, and discussions of the statistical theory of motifs and methods based on the hypergeometric distribution. Much material has been clarified and reorganized.

    The book is written so as to appeal to biologists and computer scientists who wish to know more about the statistical methods of the field, as well as to trained statisticians who wish to become involved with bioinformatics. The earlier chapters introduce the concepts of probability and statistics at an elementary level, but with an emphasis on material relevant to later chapters and often not covered in standard introductory texts. Later chapters should be immediately accessible to the trained statistician. Sufficient mathematical background consists of introductory courses in calculus and linear algebra. The basic biological concepts that are used are explained, or can be understood from the context, and standard mathematical concepts are summarized in an Appendix. Problems are provided at the end of each chapter allowing the reader to develop aspects of the theory outlined in the main text.

    Warren J. Ewens holds the Christopher H. Brown Distinguished Professorship at the University of Pennsylvania. He is the author of two books, Population Genetics and Mathematical Population Genetics. He is a senior editor of Annals of Human Genetics and has served on the editorial boards of Theoretical Population Biology, GENETICS, Proceedings of the Royal Society B and SIAM Journal in Mathematical Biology. He is a fellow of the Royal Society and the Australian Academy of Science.

    Gregory R. Grant is a senior bioinformatics researcher in the University of Pennsylvania Computational Biology and Informatics Laboratory. He obtained his Ph.D. in number theory from the University of Maryland in 1995 and his Masters in Computer Science from the University of Pennsylvania in 1999.

    Comments on the First Edition. "This book would be an ideal text for a postgraduate course.[and] is equally well suited to individual study.. I would recommend the book highly" (Biometrics). "Ewens and Grant have given us a very welcome introduction to what is behind those pretty [graphical user] interfaces" (Naturwissenschaften.). "The authors do an excellent job of presenting the essence of the material without getting bogged down in mathematical details" (Journal. American Staistical. Association). "The authors have restructured classical material to a great extent and the new organization of the different topics is one of the outstanding services of the book" (Metrika).

    Download

    Statistics and Probability for Engineering Applications, First Edition-E-Book

    Publisher Newnes
    Author(s) William DeCoursey
    ISBN 0750676183
    Release Date 01 January 2003
    More than ever, American industry especially the semiconductor industry is using statistical methods to improve its competitive edge in the world market. It is becoming more imperative that graduate engineers have solid statistical know-how, yet engineers in industry typically are not well-prepared to use statistics and they are fuzzy about how to apply statistical tools and techniques. This valuable reference makes statistical methods easier and more accessible to engineers.
    Although the book can be read sequentially, like a normal textbook, it is designed to be used as a handbook, pointing the reader to the topics and sections pertinent to a particular type of statistical problem. It contains the following features:
    * Covers all major topics treated in a standard college engineering statistics course, but minimizes the mathematical derivations and focuses on practical applications
    * Uses real data sets/case studies taken from electronics, electrical engineering, and other engineering fields, such as mechanical and chemical engineering
    * Contains numerous software examples using the powerful statistical functions of Excel

    Download

    Biostatistical Methods in Epidemiology: E-Book

    Download

    Biostatistics, Second Edition: A Guide to Design, Analysis and Discovery- E-book

    Biostatistics, Second Edition: A Guide to Design, Analysis and Discovery.

    • Publisher: Academic Press
    • Number Of Pages: 528
    • Publication Date: 2006-12-14
    • Sales Rank: 766479
    • ISBN / ASIN: 0123694922
    • EAN: 9780123694928
    • Binding: Hardcover
    • Manufacturer: Academic Press
    • Studio: Academic Press
    • Book Description:

      Today, mathematics, biology, medicine, and statistics are closing the interdisciplinary gap in an unprecedented way and many of the important unanswered questions now emerge at the interface of these disciplines. Now in its Second Edition, this user-friendly guide on biostatistics focuses on the proper use and interpretation of statistical methods. This textbook does not require extensive background in mathematics, making it user-friendly for all students in the public health sciences field. Instead of highlighting derivations of formulas, the authors provide rationales for the formulas, allowing students to grasp a better understanding of the link between biology and statistics. The material on life tables and survival analysis allows students to better understand the recent literature in the health field, particularly in the study of chronic disease treatment. Biostatistics now includes a companion website to demonstrate the different applications of computer packages for performing the various analyses presented in this text.
      * Includes access to a companion website with further examples and a full explanation of computer packages
      * Over 40% new material with modern real-life examples, exercises and references
      * New chapters on Logistic Regression; Analysis of Survey Data; and Study Designs
      * Introduces strategies for analyzing complex sample survey data
      * Written in a conversational style more accessible to students with real data

    Download

    Wednesday, 6 June 2007

    The Cell - A Molecular Approach-E Book, The Cell - A Molecular Approach Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc. ; c2000

     

     

     

     

    The Cell - A Molecular Approach

    Cooper, Geoffrey M.

    Sunderland (MA): Sinauer Associates, Inc. ; c2000

    Read This Book Full Version

    C. elegans-Ebook, Riddle, Donald L.; Blumenthal, Thomas; Meyer, Barbara

    C. elegans II

    Riddle, Donald L.; Blumenthal, Thomas; Meyer, Barbara J.; Priess, James R., editors.

    Plainview (NY): Cold Spring Harbor Laboratory Press ; c1997

    Read This Book Full

    Cancer Medicine- Ebook, Kufe, Donald W.; Pollock, Raphael E.; Weichselbaum, Ralph R.; Bast, Robert C., Jr.; Gansler, Ted S.; Holland

    Kufe, Donald W.; Pollock, Raphael E.; Weichselbaum, Ralph R.; Bast, Robert C., Jr.; Gansler, Ted S.; Holland, James F.; Frei III, Emil, editors. Hamilton (Canada): BC Decker Inc. ; c2003

    Read This book Full

    Biochemistry Ebook Berg, Jeremy M.; Tymoczko, John L.; and Stryer, Lubert. New York: W. H. Freeman and Co. ; c2002

    LUBERT STRYER is currently Winzer Professor in the School of Medicine and Professor of Neurobiology at Stanford University.

    Read This Book Full

    Basic Neurochemistry: Molecular, Cellular, and Medical Aspects-Ebook

    Siegel, George J.; Agranoff, Bernard W.; Albers, R. Wayne; Fisher, Stephen K.; Uhler, Michael D., editors

    Philadelphia: Lippincott,Williams & Wilkins ; c1999

    Read This Book

    Approved Lists of Bacterial Names-Ebook

    The Approved Lists of Bacterial Names was published in the International Journal of Systematic Bacteriology (IJSB 30: 225-420, 1980) and reprinted in book form to provide for the requirements of the Bacteriological Code (1976 Revision) in initiating a new starting date for bacterial nomenclature, 1 January 1980.

    Read This Book

    PCR Protocol

    20-mer Polymerase Chain Reaction Procedure (for MJ Research Thermal Cycler)
    Caution: When the main power to the Unit has been switched off, allow at least 5 sec before switching on again. Under no circumstance should the Unit be set to "To printer and Ram."
    1. Turn on the PHC-2 and circulator at least 20 min before an experiment.
    2. Position the sample PRT (Pretinum resistance thermometer) in one of the center holes in the block.
    3. Press the <PAGE> key to move to Unit display Page 1.
    4. Edit the program or pick up a program if the program has been set.
      Reaction time for Taq I Polymerase reaction of cotton probe DNA.
      Denature 93 C 30 sec
      Anneal 55 C 60 sec
      Extend 72 C 120 sec
      * Total cycle : 30
    5. Heat a 0.7 ml MFT containing reaction mixture for 5 min at 95 C to denature the DNA completely.
      Reaction mixture
      10X Reaction buffer 5 �l
      dNTP 0.2 mM (4 �l)
      Primer 1 100 pM (2 �l) from 50nM,100 �l TE
      Primer 2 100 pM (2 �l) "
      Template DNA 250 ng
      H2O to a final volume 50 �l
    6. Positioned the MFT containing DNA sample and add 2.5 unitTaq I polymerase (0.5 �l: 5 unit/�l; Perkin Elmer Cetus). To withdraw the required amount of Taq I polymerase from the stock, spin the stock tube 10 sec at 4 C just before or dilute the enzyme with H2O and withdraw the diluted enzyme.
    7. Overlay the reaction mixture with 100 �l of light mineral oil.
    8. Press the <RUN> key to start the program.
    9. Withdraw the sample of the amplified DNA from the reaction mixture and analyze it by gel electrophoresis. If necessary, remove the oil by extraction with 150 �l chloroform. Transfer aqueous phase, forming a micelle near the meniscus, with a P-20 to a new MFT.
    MJ Research thermal Cycler: 10-mer PCR for amplification of random genomic DNA fragments of Cotton
    1. Turn on the thermor reactor (MJ Research, model PTC 100).
    2. Edit (or choose a program if it has been set up) PCR Program.
      Tdenature = 95 C, 5 min; 1 cycle.
      Tdenature = 94 C, 30 sec,
      Tanneal= 38 C, 1 min, 45 cycle
      Textension= 70 C, 2 min;
      Textension=5 min; 1 cycle.
      T4C=4C;hold forever.
    3. Prepare reaction mixture and template cotton DNA.

    * Enzyme should be kept in ice or table-top freezer. 10X reaction buffer, dNTPand primers should be kept in ice after thawing. DNA in H2O is not sensitive as much as others. However, the DNA solution should not be kept at room temp for longer than 15 min.

    1. Number on the top of 0.5ml-microfuge tubes (MFT).
    2. Prepare reaction mixture (for example of 25 �l reaction mixture)
      1X conc.
      2.5 �l of 10X reaction buffer (Promega) -
      1.5 �l of 25 mM MgCl2 (Promega) 2 mM
      2 �l of 10 mM dNTP (Promega) 0.2 mM
      (or mixture of 0.5 �l of four 10 mM dATP, dCTP, dGTP, dTTP)
      2 �l of 2.5 mM random primer (Operon) 0.2 mM
      Template DNA 10-30 ng (20 ng is opt.)
      DNA+H2O 18 �l
    3. After tapping or voltexing the reaction mixture with DNA in 0.5ml MFT briefly, spin (10 K, 5 sec).
    4. Run the PCR program. While denaturing the DNA at 95 C for 5 min, prepare the Taq polymerase in H2O.

      For 25 �l reaction mixture in a 0.5 ml MFT, Taq polymerase 0.3 �l (1.5 Unit; 0.5 Unit/�l) dissolving in H2O 2.5 �l

    5. While the PCR cycle is processing at Td=94 C, 30 sec (1st stage and 1st of 45 cycle), open the top of MFTs.
    6. When the transition stage starts (between Td and Ta) of the 1st cycle, pause the cycle (push a decimal point button) and add a mixture of Taq polymerase (2.8 �l/tube) with EDP (25 �l liquid end) as quick as possible. Do not touch the top of reaction mixture in the tubes. Drip the Taq enzyme mixture at a distance of 1-2 mm from the mixture surface. If touched on the reaction mixture with MFTs, use a fresh tip.
    7. Add 30 �l sterile mineral oil on the top of mixture in MFTs with EDP as quick as possible and unpause the cycle (push decimal point button). It takes about 5 hr 30 min to 5 hr 45 min to finish the whole cycle. If overnight is necessary after the cycle, set 4 C (time holding) after final Te.
    8. After the cycle, add 2.5 �l of 10 X tracking dye, tap the tubes to mix the dye and bottom phase, spin (5K, 5 sec).
    9. Loading the sample (10 �l/lane) on a gel (1.4 % agarous gel is usually good). Using a two- ladder gel (2 combs) is working good.
    10. Run the gel (160 volt, 1 hr) until the 500 bp-dye reachs to the bottom of the gel.
    11. Take a picture.

    GeneAmp 9600(Perkin Elmer Cetus): 10-mer PCR for amplification of random genomic DNA fragments of Cotton

    1. Turn on the thermal cycler (GeneAmp 9600).
    2. Edit (or choose a program if it has been set up) PCR Program (Math 4).
      Tdenature = 95 C, 2 min; 1 cycle.
      Tdenature = 94 C, 30 sec,
      Tanneal= 38 C, 1 min, 45 cycle
      Textension= 70 C, 2 min;
      Textension=5 min; 1 cycle.
      T4C=4C;hold forever.
    3. Prepare reaction mixture and template cotton DNA.

      * Enzyme should be kept in ice or table-top freezer. 10X reaction buffer, dNTPand primers should be kept in ice after thawing. DNA in H2O is not sensitive as much as others. However, the DNA solution should not be kept at room temp for longer than 15 min.
      1) Number on the top of 200 �l thin-wall tubes.
      2) Prepare reaction mixture (for example of 25 �l reaction mixture)
      1X conc.
      2.5 �l of 10X reaction buffer (PerkinElmer) -
      2 �l of 10 mM dNTP (PerkinElmer) 0.2 mM
      (or mixture of 0.5 �l of four 10 mM dATP, dCTP, dGTP, dTTP)
      2 �l of 2.5 mM random primer (Operon) 0.2 mM
      Template DNA 10-30 ng (20 ng is opt.)
      DNA+H2O 17 �l
      AmpliTaq 0.3 �l (1.5 Unit)
      (Voltex the AmpliTaq tube briefly before aliquoting the enzyme)

    4. Secure the cap with the roller tightly.
    5. Voltex the reaction mixture with DNA in the tube briefly. Spin the tubes with adaptors (10 K, 5 sec), if necessary.
    6. Start the PCR program. It takes about 4 hr to 4 hr 10 min to finish the whole cycle. If overnight is necessary after the cycle, set 4 C (time holding) after final Te.
    7. After the cycle, add 2.5 �l of 10 X tracking dye/25 �l reaction tube, voltex the tubes briefly to mix, spin (10K, 5 sec) if necessary.
    8. Loading the sample (10 �l/lane) on a gel (1.4 % agarous gel is usually good). Using a two-ladder gel (2 combs) is working good.
    9. Run the gel (160 volt, 1 hr) until the 500 bp-dye reachs to the bottom of the gel.
    10. Take a picture.

    TA cloning or Subcloning of PCR Products Protocol

    TA Subcloning of PCR Products

    This procedure is adapted from D. Marchuk, M. Drumm, A. Saulino, and F.S. Collins Nuc. Acids. Res. (1991) 19:1154.
    CONSTRUCTION OF T-VECTOR
    1. suspend 10ug pUC 19 in:
      • 4.0ul 10X reaction buffer (we use Bo. Mann. buffer A)
      • 2.0ul (20U) Sma I
      • X ul dwater to a total vol. of 40ul
        Incubate at 30 (not 37) degrees for 1 hour. This is easier if done in a 0.4ml tube in a thermal cycler.
    2. Heat to 70 degrees for 15 min. to kill the enzyme
    3. Bring to 100ul w/ water (add 60ul).
    4. Extract w/ phenol, phenol/chloroform and then chloroform.
    5. add 9ul 3M sodium acetate.
    6. ppt. in ETOH, wash with 70% ETOH (be careful with the pellet!).
    7. Dry in spin vac at room temp (do not use heater!).
    ********************T-TAILING THE VECTOR******************
    At this point, it is assumed that there has been 80% recovery of the cut plasmid DNA.
    1. Resuspend the plasmid DNA in 63ul water (conc approx. 130ng/ul)
    2. To the resuspended plasmid add:
      • 10ul 10X PCR buffer (standard cetus stuff, no MgCl)
      • 20ul 10mM dTTP [2mM final]
      • 6ul 25mM MgCl2 [1.5mM final]
      • 1ul Taq polymerase (Cetus amplitaq 5U/ul)
      • ______
      • 100ul total volume.
    3. Incubate for 3 hours at 70 degrees C.
    4. Extract with Phenol, Phenol/chloroform, chloroform.
    5. Extract twice with ether (so I'm paranoid!)
    6. add 75ul 2M **ammonium** acetate (assuming 75ul recovery from extractions).
    7. Add 150ul isopropanol. Spin 20mins in microfuge at full speed at 4 degrees.
    8. Wash with 70% ETOH 9 Dry pellet in spin vac and store at -20 degrees until use.
    TREATMENT THE PCR PRODUCTS
    " If you can see it, you can clone it".
    1. Add an equal volume of chloroform (*NO* IAA) to the PCR reaction and spin 1-2 minutes in microfuge at RT.
    2. Remove the oil which is now on the ****BOTTOM***.
    3. Spin again for two minutes and remove the last little bit of oil from the bottom. You will know when you have gotten it all when you see the interface in the pipette tip. It is important that all the oil be removed otherwise subsequent procedures will be very difficult.
    4. Add 100ul 4M ammonium acetate, vortex, and then add 200ul isopropanol.
    5. Centrifuge 20min at 4 degrees, wash in 70% ETOH.
    6. Dry in speed vac.
    7. Resuspend the DNA in 8-10ul TE, add loading buffer and load onto a 4% Nusieve (TAE) agarose gel. Run until the desired band is well separated. The more DNA in the band, the easier it is to subclone.
    8. Cut out the band. Minimize the exposure of the gel (and you!) to short wave UV
    LIGATION OF PCR PRODUCTS TO T-VECTOR
    1. Heat the gel containing the PCR fragment to 65C for 10 minutes, place in a 37C water bath or block and add to a separate tube (also at 37C):
      • 10 ul gel
      • 4ul 5X ligase buffer (commercial buffer that comes with BRL T4 ligase)
      • 4ul water
      • 1ul vector (25-50ng)
      • 1ul ligase
      • Incubate at 12C overnight.
    2. Heat the mixture to 68 degrees for 5 minutes and add 100ul water.
    3. Extract with phenol, phenol/chloroform, and chloroform. These steps are to remove the agarose.
    4. Add 10ul 3M NaAcetate and precipitate with ethanol.
    5. Wash the pellet in 70% ETOH, dry in the speed-vac. Resuspend in 5ul of water just prior to transformation.
    *Transformation - We usually use electroporation into XL-1 blue cells. You need cells that can achieve at least 1 x 10^7 transformants per ug of DNA if a CaCl based protocol is used.

    *Storage: The T-vector should be stored at -20C at all times. When stored in dry form, the T-overhangs will last longer (I don't know how long yet). In solution, it lasts at least a couple of weeks at - 20C.

    *Enzymes - The batch of SmaI that is used is particularly critical. Some are contaminated with an endonuclease that removes a few bases from the cloning site. The batch of smaI should be checked before it is used to cut vector for cloning purposes. If bluescript is used, EcoRV can be substituted for sma I.

    txpljfg@uabcvsr.cvsr.uab.edu

    Cloning of Blunt-end PCR Fragments Protocol

    Direct Cloning of Blunt-end PCR Fragments

    BioTechniques 13:613
    • Phenol extract the PCR product
    • Ethanol precipitate
    • Treat for 1hr at 37C with 10 units of T4 DNA polynucleotide kinase and 10 units T4 DNA polymerase I(NEB) in a 100ul reaction volume containing 50mM Tris-HCl pH7.5, 10mM MgCl2, 1mM DTT, 50 ug/ml BSA, 1mM ATP, 200uM each dNTP.
    I run the entire thing out on a 1.3% agarose TAE gel, cut a trough in front of the band, pour in some 0.7% LMP agarose(BRL), run the product into it and excise.

    The PCR product in the LMP can be used for ligations directly, without purification. The ligations take place at room temp on the benchtop. I prepare the vector with minimal digestion (~2hr) then treat it with shrimp alkaline phosphatase(USB). I usually prepare a stock of this vector to have on hand, so I know it is good and will have a low background. You may also want to try using an EcoRV cut vector instead of a Sma cut vector.

    • Remove the oil with Diethylether.
    • To 40 ul of the PCR reaction add 50 ul of H2O. Add 10 U T4 PNK, 10 U klenow, ATP (to 1 mM) and some more dNTPs (usually 4 ul of 1.25 mM...whatever) and icubate at 37 oC for 30 min.
    • Phenol/chloroform extract
    • chloroform/IAA extrac
    • EtOH precipitate
    you can then just "shotgun" clone this DNA. However, if you have several non-specific bands then you may want to gel purify the fragment first.

    One point to note....

    We have given up blunt end cloning into Sma I sites where possible. Several people have reported problems with Sma I cut DNA. By choice we clone into EcoRV sites.

    Agarose Gel Electrophoresis-Protocol

    Agarose Gel Electrophoresis

    Caution: wear gloves because of Etedium Bromide(Cancer Causing).
    1. Dilute the 10X running buffer (TBE with EtBr) to 1X. Calculate the amount of the 1X buffer in gel tray(s). Add appropriate amount of agarous (for 5 mm standard gel; 0.7 % or 1 %) in the buffer, melt in the microwave oven. Pour the melting agarous (appr. 40-50 C) into a graduate cylinder, add 1X buffer up to the pre-determined volume, pour back into the flask to mix, and fill the gel tray (watch out bubble). Let set at least one hour to harden.
       Gel size (W X L) Agarous (Rec)  
      Gel tray (cm X cm) Owl Rec. Buffer (L) 1.0 % 0.7 %

      A3 20 X 40 600 400 3.0 L 4.0 2.8

      B2 12 X 14 130 100 0.5 L 1.0 0.7

      C1 7.6 X 5.1 30 20 0.1 L 0.3 2.7


    2. Fill an electrophoresis chamber with 1X running buffer until the gel is covered by a couple of millimeters.
    3. Adjust DNA concentration of sample (appr. 5 to 8 �g genomic DNA; 0.5 �g plasmid DNA per lane).
    4. Load the DNA samples (with 1/10 volume of tracking dye and heat shocked for 5 min at 65) carefully with a Pipetman P20 by slowly expelling the solution into a well, with the pipet tip slightly below the top of the well. Do not hold the pipet too far in the well: the sample will sometimes come out the bottom. Try not to plug the pipet tip against the side of the well either: the sample will usually squirt out when there gets to be enough pressure from the pipetman.
    5. Load the appropriate marker DNA flanking the sample lanes.
    6. Close the lid on the electrophoresis kit. Always run the DNA toward the red (+) terminal. Electrophoresis the gel at 11 volts overnight or 100 volts for a couple of hours.

      Note: the size of DNA to be separated needs to be matched to the agarous concentration:

      Agarose %	Range of separation
      0.3 60 - 5 kb
      0.6 20 - 1 kb
      0.7 10 - 0.8 kb
      0.9 7 - 0.5 kb
      1.2 6 - 0.4 kb
      1.5 4 - 0.2 kb
      2.0 3 - 0.1 kb

    X-Gal Protocol (Promega Biotech)

    X-Gal Protocol

    Caution: X-gal stock solution contains Dimethylformide

    X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) turns blue when incubated in the presence of b-glactosidase. This gene is on several of the cloning plasmids (especially, on the pUC series and lGT11 vectors). When an inserted piece of DNA is placed in the correct restriction site, the lacZ gene is interrupted and the colony does not turn the media blue (colony we want). Be sure to run controls.

    1. Make 13X100 tubes with 2.5 ml LB and 0.8 agar by melting then dispensing it into the tubes and autoclaving it for only 5 min.
    2. Use the tubes while hot or re-melt briefly and hold at 42 C.
    3. Add 20 �l 20 mg/ml IPTG (filter sterilized in H2O), 50 �l 20 mg/ml X-gal (in Dimethylformide), and 1 �l Crb solution (100 mg/ml).
      Note: Dimethylformide seems to melt plastic so make the stock in glass or PP (or PA) oakridge tubes.
    4. Add the transformed cells (try to get about 200 CFU in up to 250 �l). Vortex and overlay on a CA plate containing the appropriate antibiotic (usually Crb).
      * 2.5 ml is a little tricky to overlay neatly: do not replace cap, tilt the plate to get uniform coverage of the overlay.
    5. Let the agar solidify then incubate at 37 C until the blue color develops.
    6. Pick the colorless colonies to media with the correct antibiotic and verify the insert by mini plasmid preparations or colony hybridization.

    Transformation of E.Coli Protocol

    Transformation DNA fragments (or plasmid DNA) into competent E. coli
    * Caution: Use aerosol protecting tips if selection of transformed cells is not based on X-gal strategy.
    1. Remove competent cells (E.coli DH5aTM from GIBCO BRL) from -70 C freezer; thaw on wet ice.
    2. Place four 15-ml modified polystylene tubes (PST; Corning disposable sterile centrifuge tube) on ice.
    3. Gently mix cells (tapping with fingers), then aliquot 50╩Ál competent cells into each of chilled the 15 ml PST.
    4. Add 1 �l of recombinant DNA sample (1 - 2 �g DNA) to the competent cells by moving the pipette through the cells while dispensing. Gently tap tubes to mix.
      * To prepare the recombinant DNA sample: if desired, wash the recombinant DNA of the interest (should be less than 10 kb which is usually digested with a restriction enzyme) twice in ultrafree 1.5 ml MFT by centrifugation (5000 rpm, 5 min, 4 C) by letting spin down to dead stop volume (ca. 5 to 20 �l).
      * Ultrafree MC polysulfone; 100,000 NMWL; Nihon Millipore Kogyo K.K. Yonezawa, Japan.
    5. Incubate cells on ice for 30 min.
    6. Heat-shock cells 45 sec in a 42 C water bath: Do NOT shake.
    7. Place on ice for 2 min
    8. Add 0.95 ml of room temperature SOC.
    9. Add 5, 10, 20, 50, 100, and 200 �l (duplicate) of the diluted DNA sample into 2.5 ml of 0.8 % LB at 42 C. Do this step and the next step within 5 min (-42 C).
      * Dilution of control DNA (pUC19) are duplicate of 5, 10, 50, 100, 200 �l of the diluted DNA samples. If size of the tranforming DNA is big size e.g. >5 Kb, start 50 �l diluted DNA sample into overlay medium.
    10. Do overlay on LB containng Carbenicillin plates (filter sterilized Crb 100 �g/ml or Amp 100 �g//ml).
    11. Incubate at 37 C.

    How to Prepare of Competent Cells for transformation? (protocol)

    Preparation of Competent Cells (from PMB)

    1. Inoculate a couple of 20 ml LB broth in a 125 ml flasks with 0.5 ml of an overnight culture of the recipient strain.
    2. Shake at 37 C until O.D. 600=0.13 - 0.15 (1 - 2 hours). Measure by taking some of the broth out with a sterile DPTP and reading in a plastic cuvette (discard cells afterward): If flanking arm flasks are accessible, use them for measuring O.D.
    3. Spin the cells in sterile Oak Ridge Tube (5 K, 5 min, 4 C) and resuspend in 1 ml PMB #A. Then add another 9 ml PMB #A and spin the cells again (5 K, 5 min, 4 C).
    4. Decant and resuspend in 1 ml PMB #B. Then add 9 ml more PMB #B.
    5. Let sit on ice for 30 min, spin as above and resuspend in 2 ml PMB #B, add 0.2 ml glycerol and 30 �l DMSO. Use fresh or freeze in dry ice/EtOH.

    PMB #A
    1X 200 ml 1X
    10 mM MOPS pH 7.0 210 mg Free acid
    230 mg Na salt
    10 mM Rubidium chloride 240 mg
    Autoclave and store at 4 C
    PMB #B
    1X 200 ml 1X
    10 mM MOPS pH 6.5 350 mg Free acid
    85 mg Na salt
    50 mM Calcium chloride 1.12 g
    10 mM Rubidium chloride 240 mg
    Autoclave and store at 4 C

    Antibiotic selections concentrations?

    Antibiotics should be added to lower than 60 C broth, or filter sterilized: See note. "C' refers to chromosomal resistance: "P" plasmid based resistance, "FS" filter sterilization required. Temperature is required for storage.
    Concentration (�g/ml)

    Antibiotics for Pseudomonas, E. Coli, Agrobacterium, etc...
    Ampicillin 1000 (C/P) 50 (P) Stock: 25 mg.ml Na salt:FS: Store at -20 C, carbenicillin is more stable than Amp.
    Carbenicillin 1000 (C/P) 50 (P) 100 mg/ml: FS: -20 C
    Chlroramphenicol 500 (C) 30 (C/P) 170 �g/ml for plasmid amplification 34 mg/ml in EtOH: -20 C
    D-cycloserine 200 (C) Add dry after autoclaving
    Erythromycin 200 (C) 25 mg/ml EtOH: -20 C
    Gentamycin 10 (C/P) Add dry after autoclaving
    Geneticin 200 for fungi Same as kanamycin in mode of action and preparation
    Hygromycin 250 for fungi 25 mg/ml: FS: -20 C
    Kanamycin 200 (P) 50 (P) 25 mg/ml: FS: -20 C
    Kasugamycin 200 (C) 20 mg/ml: FS: -20 C
    Mercuric chloride 40(C/P) 10 �g/ml if in minimal media, 40 mg/ml in EtOH: -20 C
    Nalidixic acid 1000 (C) 20 (C) 100 mg/ml in H2O, add 6 N NaOH until dissolved: 4 C
    Neomycin 200 (P) 50 (P) 25 mg/ml: FS: -20 C
    Rifampicin 200 (C) 200 mg/ml make fresh in Methanol
    Spectinomycin 200 (C/P) 25 (P) 20 mg/ml: FS: -20 C
    Streptomycin 200 (C/P) 25 (P) 20 mg/ml: FS: -20 C
    Sulfamethoxazole 500(C) Add dry after autoclaving
    Triomethoprim 500 (C) Add dry after autoclaving
    Tetracycline 20(C)50(P) 15 (P) 12.5 mg/ml 50 % EtOH: -20 C

    How to make stock solutions? BSA, EtBr, 0.5 M EDTA, 0.5 M Na3EDTA, Polyethylene Glycol, Perchloric acid, 1M NaHPO4, Tris 1 M, Treacking Dye, Glycerol Solution, RNase A and RNase T1

    Stock Solution
    BSA
    Stock BSA from BRL comes as 50 mg/1000 �l. Add 4 ml of H2O for a final concentration of 10 mg/ml. Store at -20 C.
    EtBr (10 mg/ml)
    Dissolve 100 mg EtBr in 10 ml H2O. Stir well and store at 4 C in an amber bottle.
    0.5 M EDTA (pH 8.0): 500 ml
    Dissolve 95.05 g of Na2EDTA in appr. 400 ml H2O. Adjust pH to 8.0 with 6 N NaOH. Then bring volume to 500 ml and autoclave.
    0.5 M Na3EDTA: 1L
    Add 186.2 g (?) to 900 ml H2O. Adjust pH with conc HCl (or NaOH ?) to 8.0, and bring up to 1 L.
    5 M LiCl 50 mM MOPS (pH 8.0)
    Conc
    5 M LiCl 21.2 g
    50 mM MOPS 1.05 g
    H2O up to 100 ml
    Adjust pH 8.0 with NaOH. Store at 4 C.
    50 % PEG
    Add 100 g PEG (Polyethylene Glycol, appr. MW 8.000) to 50 ml H2O and stir for about one hour. Then bring up to 200 ml with H2O.
    3 N Perchloric acid
    Dilute 49.2 ml of perchloric acid up to 200 ml with H2O in a hood. Stable at room temperature for several month. Treat with care.
    1 N NaOH
    Dissove 40 g of NaOH in 1000 ml H2O.
    1M NaHPO4
    Stock
    NaH2PO4.H2O (monobasic monohydrate) 47.5 g
    Na2PO4.7H2O (dibasic 7-hydrate) 42.2 g
    H2O to 500 ml
    10 M NH4Ac
    Add 400 ml H2O to a 1 Kg bottle NH4Ac, dissolve, and adjust to 1300 ml.
    Tris 1 M pH 8.0
    Stock
    Tris-HCl 88.8 g 177 g 355.2 g
    Tris-Base 53.0 g 106.0 g 212.0 g
    H2O 1L 2 L 4 L
    Tris 1 M pH 7.5
    Stock
    Tris-HCl 127 g 254 g 508 g
    Tris-Base 23.6 g 47.2 g 94.4 g
    H2O 1 L 2 L 4 L
    76 % EtOH 10 mM NH4Ac
    Conc. Stock Volume
    75 % 95 % EtOH 80.0 ml
    0.2 M 5 M NH4OAc 0.2 ml
    H2O 19.8 ml
    5 M NH4Ac stock = 38.55 g/100 ml
    76 % EtOH 0.2 M NaAc
    Conc. Stock Volume
    76 % 95 % EtOH 160 ml
    0.2 M 3 M NaAc 13.33
    H2O 26.67
    3 M NaOAc stock = 40.83 g/100 ml
    0.2 N NaOH 0.6 M NaCl
    1L 2L 3L 4L
    NaOH 8.0 g 16.0 g 24.0 g 32.0 g
    NaCl 35.04 g 70.08 g 105.2 g 140.2 g
    0.5 M Tris (pH 7.5) 1.5 M NaCl
    1L 2L 3L
    Tris-HCl 63.5 g 127.0 g 190.0 g
    Tris-Base 11.8 g 23.6 g 35.4 g
    NaCl 87.6 g 175.0 g 262.8 g
    25 mM NaHPO4
    1M NaHPO4 100 ml
    H2O to 4 L
    10 X Treacking Dye
    Conc.
    50 mM Tris pH 8.0
    50 mM Na3EDTA
    25 % Glycerol
    5 % Ficoll
    0.1 % Xylenol Cyanol
    0.1 % Bromophenol Blue
    1 % SDS
    Glycerol Solution
    Glycerol 650 ml
    1 M MgSO4 100 ml
    1 M Tris (pH 8) 25 ml
    H2O up to 1 L
    Mix well and autoclave for 15 min at 121 C.
    0.1 M Spermidine Solution
    Dissolve 255 mg spermidine (Sigma #S2501) in H2O to a final volume of 10 ml. Store at -20 C.
    Saline-MOPS
    1X 1L 1X
    0.85 % NaCl 8.5 g
    50 mM MOPS 6.3 g MOPS free acid
    4.7 g MOPS Na salt
    pH 7 autoclave and store at room temperature.
    Proteinase K - 10 mg/ml in TE
    Weigh out 10 mg proteinase K (Boeringer Manheim #161 497) and dissolve in 1 ml TE. The solution can be used immediately or aliquoted and stored at -20 C
    RNase A and RNase T1
    Dissolve 100 mg RNase A (bovine pancrease: Sigma #R4875), if desired. add 5,000 units of RNase T1 together, in 10 ml of 10 mM Tris 15 mM NaCl. Boil for 15 min and allow to cool slowly to room temperature. Distribute 1 ml aliquote into 1.5 ml MFT, and store at -20 C. If

    Buffer Recipes: 10X TE,TrisEDTA, TBE Buffer, TAE Buffer, DNA Extraction bufferReaction Stop Buffer, STE, SSC, Hybridization Buffer, Oligo Buffer, Klenow buffer, Reaction buffer,

    Buffer Formula (required precision; 2 %)

    Always to try to prepare buffer solution at the temperature and concentration before planing to use during the experiment. Stock solutions are acceptable as long as pH adjustment made after temperature and concentration adjustment. Also good buffers, e.g., Tris and Phosphate, are stable for a long time period.

    10X TE (10:2)

    1X 1L 10X
    10mM Tris-HCl 8.9 g Tris-HCl
    5.3 g Tris-Base
    2 mM Na3EDTA 7.4 g
    pH 8 at 25 C. Mix and store at 4 C. Periodically dump the 1X TE and make up the fresh 1X TE from 10X stock.

    10X TE (10:1)

    1X 1L 1X 1L 10X
    10mM Tris-HCl 1.06 g Tris-HCl 10.6 g
    0.39 g Tris-Base 3.94 g
    1mM Na2EDTA 0.475 g 4.75 g
    pH 8 at 25 C. Mix and store at 4 C.

    10X TE (50:50)

    1X 1L 10X
    50 mM Tris-HCl 44.4 Tris-HCl
    26.5 Tris-Base
    50 mM Na3EDTA 186 g
    Store at 4 C.

    TE (25:10) 50 mM Glucose

    1X 0.25 L 1X
    25 mM Tris-HCl 0.55 g Tris-HCl
    0.33 g Tris-Base
    10 mM Na3EDTA 0.92 g
    50 mM Glucose 2.25 g
    Store at 4 C.

    50 mM Tris pH 8.0 10 mM EDTA

    50 mM 1 M Tris pH 8.0 5 ml
    1 mM 0.5 M EDTA 2 ml
    H2O up to 100 ml
    Store at 4 C.

    1 M Tris pH 8.0, 1.5 M NaCl

    1X 1L 1X 2L 1X
    1M Tris-HCl 88.8 g Tris-HCl 178 g
    53.0 g Tris-Base 106 g
    1.5 M NaCl 87.7 g 175 g
    Store at 25 C (room temperature is OK)

    1 M Tris pH 7.4, 1.5 M NaCl

    1X 1L 1X 2L 1X
    1M Tris-HCl 132.2 g Tris-HCl 264.4 g
    19.4 g Tris-Base 38.8 g
    1.5 M NaCl 87.7 g 175 g
    Store at 25 C (room temperature is OK)

    0.5 M Tris pH 7.5, 1.5 M NaCl

    Conc. 1 L 2L 3 L
    0.5 M Tris Tris-HCl 63.5 g 127.0 190.0
    Tris-Base 11.8 g 23.6 g 35.4 g
    1.5 M NaCl 87.6 g 175.0 g 262.8 g
    Store at 25 C (room temperature is OK)

    10 mM Tris pH 7.5, 15 mM NaCl


    Conc. Stock Volume
    10 mM 1 M Tris pH 7.5 100 �l
    15 mM 5 M NaCl 30 �l
    H2O to 10 ml
    Store at 4 C.

    10X TBE Buffer (pH 8)

    1X 1L 10X 4L 10X
    89 mM Tris-Base 108 g 432 g
    89 mM Boric acid 55 g 220 g
    2 mM Na2EDTA 9.3 g 37.2 g
    EtBr (10mg/ml) 0.5 ml 2 ml
    Mix and store at room temperature without EtBr, 4 C with EtBr in a brown bottle. Use EtBr stock solution (10 mg EtBr/ml) when TBE is made.

    50X TAE Buffer (pH 8)

    1X 1L 50X 1 L 10X
    40 mM Tris-acetate 242 g Tris Base 48.4 g
    57.1 ml acetic acid 11.4 ml
    2 mM Na2EDTA 37 g 7.4 g
    EtBr (10mg/ml) 2.5 ml 0.5 ml
    Mix and store at room temperature without EtBr, 4 C with EtBr in a brown bottle. Use EtBr stock solution (10 mg EtBr/ml) when TAE is made.

    Cotton DNA Extraction buffer (pH 6)

    Conc. Stock 1L 1X
    100 mM Na2citrate.2H2O 29.4 g
    Glucose 63 g
    5 mM 0.5 M Na2EDTA 10 ml
    Na2Diethyldithiocarbamic acid 10 g
    PVP-40,000 MW 20 g
    BSA 10 g
    Adjust to pH 6.0 with HCl. Store at 4 C.

    RSB (Reaction Stop Buffer)

    Conc. Stock Volume (ml)
    10 mM 1 M Tris pH 8.0 1.0
    2 mM 0.5 M EDTA 0.4
    0.2 % 20 % SDA 1.0
    H2O 97.6
    Store at room temperature.

    STE (Sodium chloride and TE) pH 8.0

    Conc. Stock 1X 1 L 10X 1L
    10 mM 1M Tris pH 8.0 10.0 8.88 g Tris-HCl
    5.3 g Tris-Base
    1 mM 0.5 M EDTA 2.0 4.65 g Na2EDTA
    100 mM 5 M NaCl 20.0 5.84 g NaCl
    H2O up to 1 L up to 1 L
    pH 8.0 at 25 C. Store at 4 C.

    1.5 M NaCl 0.5 N NaOH

    1X 1L 1X 2L 1X
    0.5 N NaOH 20.0 g 40 g
    1.5 N NaCl 87.7 g 175 g
    Store at room temperature.

    0.2 N NaOH 0.6 M NaCl

    Conc. 1L 2L 3L 4L
    0.2 N NaOH 8.0 g 16.0 g 24.0 g 32.0 g
    0.6 M NaCl 35.04 g 70.08 g 105.2 g 140.2 g
    Store at room temperature.

    20X SSC (Adjust pH 7.0)

    1X 1L 20X 2L 4L 6L
    150 mM NaCl 175.3 g 350.6 g 701 g 1052 g
    15 mM Na3Citrate 88.3 g 176.6 g 353 g 530 g
    Mix, adjust pH to 7.0 with HCl, and store at 4 C

    25X SSC (Adjust pH 7.4)

    25X 1L 4L
    3.0 M NaCl 219 g 876 g
    0.3 M Na2Citrate 110 g 440 g
    Mix, adjust pH to 7.4 with HCl, and store at 4C.

    3 M K 5 M Ac

    1X 200 ml 1 X
    3 M KAc 29.4 g (or 20 ml of 5 M KAc)
    2 M Acetic acid 23 ml
    Dissolve the KAc in 150 ml of H2O, bring to 177 ml, and then add the glacial acetic acid. Mix and store at 4 C.

    Minimal Hybridization Buffer

    Conc. Stock 1L 2L
    10 % 50 % PEG 200 400
    7 % 20 % SDS 350 700
    0.6 X 25 X SSC 24 48
    10 mM 1 M NaHPO4 10 20
    5 mM 0.5 M EDTA 10 20
    100 �g/ml Denature Salmon Sperm 10 20
    H2O 396 792
    Aliquote 45 ml into 50 ml PPT, store at -20 C.

    Oligo Buffer


    After preparing the following stock solutions, mix A, B, and C in a ratio of 1:2.5:1.5, repectively.

    Solution A

    Stock
    2-mercaptoethanol (bME) 18 �l
    DXTPs (A, T, G) 5 �l each
    Solution O 850 �l
    Store at -20 C

    Solution O

    Conc. Stock 250 ml 100 ml
    1.47 M Tris-Base 44.52 g 17.81 g
    0.147 M MgCl2 7.47 g 2.99 g
    Adjust pH to 8.0 with conc. HCl.

    Solution B

    : 2M hepes buffer
    Conc. Stock 250 ml 100 ml
    2 M Hepes 119.15 g 47.66 g
    Adjust pH to 6.6 with 4 N NaOH. Store at 4 C.

    Solution C

    Hexamer or oligo nucleotides. Add 55 �l H2O directly to the Pharmacia bottle which contains 50 units of lyophilized hexamer. Store at -20 C.

    Klenow

    Dilute to 1 unit Klewnow fragment by adding klewnow buffer. Stock Klenow from BRL comes as 500 units in 84 �l. Dilute to 1 unit by adding 420 �l of klenow buffer to the 500 unit/84 �l stock. Store - 20 C.

    Klenow buffer (250 ml)

    Conc Stock
    7 mM Tris-HCl 0.211 g
    7 mM MgCl2 0.355 g
    50 mM NaCl 0.730 g
    50 % Glycerol 125 ml
    Add the above to about 80 ml H2O. Stir to dissolve. Adjust volume to 250 ml with H2O. Store at -20 C.

    REact buffer (See Restriction enzyme buffer formula)

    10 X REact buffer 2 : for, e.g. Hind III and Sst 1 (5 ml)

    1 X Conc. Stock
    500 mM 1 M Tris (pH 8.0) 2.5 ml
    100 mM 1 M MgCl2 0.5 ml
    500 mM NaCl 0.5 ml
    25 mM Spermidine(3HCl) 0.032 g
    H2O 1.5 ml
    Mix, aliquote 2 ml in screw-cap vial, store at -20 C.

    10 X REact buffer 3 : for, e.g. Eco RI (5.0 ml)

    1 X Conc Stock
    500 mM 1M Tris (pH 8.0) 2.5 ml
    100 mM 1M MgCl2 0.5 ml
    1 M 5 M NaCl 1.0 ml
    25 mM Spermidine(3HCl) 0.032 g
    H2O 1.0 ml
    Mix, aliquote 2 ml in screw-cap vial, store at -20 C.

    Science Protocols