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Lab FAQ from Roche Great information – compact format – the RAS Lab FAQS, 3rd edition Dear Valued Customer, At Roche Applied Science, we know that time is an increasingly scarce resource these days. Relevant information, available at your fingertips at the right time and in an easily accessible format can help you perform your experiments more efficiently. Based upon requests from many different customers, Roche Applied Science has updated Lab FAQS. We hope you find this new compendium of information even more useful for your laboratory work. Think of the Lab FAQS as our way of saying “Thank you” for using our reagents, kits and systems over the years. We wish you continued success in your research projects. Sincerely Roche Applied Science PS: To offer comments and/or suggestions on content, structure and/or layout of this guide, please don’t hesitate to contact Bettina Kruchen (e-mail: [email protected]) or your local representative. Your cooperation in improving Lab FAQs is highly appreciated. Acknowledgements: We thank all contributors and editors for their diligent efforts during the proofreading and production of this book. Without their help, this project would never have been realized. Editorial management 3rd edition: Critical reading of the manuscript: Doris Eisel, Design and Layout Yair Argon, University of Chicago, USA Urs W. Hoffmann-Rohrer, DKFZ , Heidelberg, Editor in Chief Hans-Joachim Degen, RAS, Germany Bettina Kruchen, Editor in Chief Ingrid Hoffmann, DKFZ, Heidelberg, Germany Paul Labaere, Scientific Advice Jürgen Kartenbeck, DKFZ, Heidelberg, Germany Stefanie Gruenewald-Janho, Scientific Advice Bernhard Korn, RZPD, Berlin/Heidelberg, Germany Oliver Seth, Scientific Advice Annette Moritz, RAS, Germany Michael Hoffmann, Scientific Advice Sabine Muench-Garthoff, RAS, Germany Doris Schmitt, Scientific Advice Attila Nemeth, University of Budapest, Hungary Jasmina Putnik, Scientific Advice Hanspeter Pircher, University of Freiburg, Germany Gudrun Tellmann, Scientific Advice Herwig Ponstingel, DKFZ, Heidelberg, Germany Hans-Jürgen Rode, Scientific Advice Hans-Richard Rackwitz, DKFZ, Heidelberg, Germany Rita Rein, Scientific Advice Hanspeter Saluz, HKI, Jena, Germany Simone Pitz, Scientific Advice Birgit Simen, University of Chicago, USA Brigitte Hloch, Scientific Advice Giulio Superti-Furga, EMBL, Heidelberg, Germany Martin Vingron, MPI, Berlin I Table of contents (general overview): 1. Working with DNA. 1 2. Working with RNA . 59 3. Working with Proteins. 81 1.1. Precautions for handling DNA . 2 2.1. Precautions for handling RNA . 60 3.1. Precautions for 1.2. Commonly used formulas . 3 2.2. Commonly used formulas . 63 handling Proteins . 82 1.3. Isolating and purifying DNA . 6 2.3. Isolating and purifying RNA . 65 3.2. Conversion from Nucleic Acids to Proteins. 88 1.4. Analyzing DNA. 9 2.4. Analyzing RNA . 68 3.3. Analyzing Proteins . 92 1.5. Cloning of DNA . 22 2.5. RT-PCR. 70 3.4. Quantification of Proteins . 98 1.6. Labeling of DNA and 2.6. Labeling of RNA . 79 3.5. Purifying Proteins . 102 Oligonucleotides . 29 2.7. References. 81 1.7. Amplification of DNA . 36 3.6. References . 112 1.8. Quantitative Real-Time PCR . 46 1.9. References . 59 Table of contents (general overview): 4. Working with Cells . 112 6. Conversion Tables 7. Addresses, Abbreviations 4.1. Precautions for handling Cells . 113 and Formulas . 163 and Index 169 4.2. Basic Information. 117 6.1. Nucleotide ambiguity code. 164 7.1. Selection of useful addresses . 170 4.3. Manipulating Cells. 123 6.2. Formulas to calculate melting 7.2. Lab Steno . 172 temperatures . 164 4.4 Analyzing Cells. 131 7.3. Abbreviations . 173 6.3. %GC content of different 7.4. Alphabetical Index . 176 4.5. References . 144 genomes . 165 6.4. Metric prefixes . 165 6.5. Greek alphabet . 166 5. Preparing Buffers 6.6. Properties of radioisotopes. 166 and Media . 145 6.7. Temperatures and Pressures. 167 5.1. Buffers . 146 6.8. Centrifugal forces . 167 5.2. Antibiotics . 160 6.9. Periodical system of elements . 168 5.3. Media for Bacteria . 161 6.10. Hazard Symbols. 168 5.4. References . 163 6.11. References. 169 II Table of contents (detailed overview): 1. Working with DNA . 1 1.5. Cloning of DNA . 22 1.1. Precautions for handling DNA . 2 Ligation with T4 DNA Ligase . .22 1.2. Commonly used formulas . 3 Dephosphorylation with Shrimp Alkaline Phosphatase . .23 Examples . .5 Modifying sticky ends to blunt ends: 5´protruding and 3´recessed ends . .24 1.3. Isolating and purifying DNA . .6 Klenow – for partial or complete filling of Perform nucleic acid isolation rapidly and efficiently . .8 3´recessed ends . .25 Sizes and weights of DNA from different organism . .8 Mung Bean Nuclease – for removing of 1.4. Analyzing DNA. 9 3´ and 5´ protruding ends . .26 Concentration and purity via OD measurement . .9 Miscellaneous . .27 Fragment Sizes of DNA Molecular Weight Markers Commonly used bacterial strains . .28 and recommended Types of Agarose . .10 1.6. Labeling of DNA and Oligonucleotides . 29 Size estimation of General Considerations . .29 – DNA fragments in Agarose MP gels . .11 Overview of different techniques . 30 – DNA fragments in Acrylamide gels . .11 Dig Labeling of Nucleic Acids . 31 Restriction Enzymes: Random Primed Labeling with Klenow . .32 – General Information . .12 Nick Translation with DNA Polymerase I and DNAse I . .33 – Buffer System . .12 3´End labeling with Terminal Transferase . 34 – Star Activity . .13 5´End labeling with Polynucleotide Kinase . .35 – Reference Materials available. .13 – Inactivation and removal . .14 – Characteristics. .14 – Cross index of recognition sequences . .19 Partial Digest . .21 1.7. Amplification of DNA . 36 1.8. Quantitative Real-Time PCR . 46 Avoiding Contamination . .36 Methods . 46 Reaction Components: Instrument-based Systems . .47 – Template . .37 Real-Time PCR Assay Formats . 48 – Primers . 38 Sequence-Independent Detection Assays . 49 – Oligonucleotides: Commonly used Formulas . 39 Sequence-specific Probe Binding Assays . 50 Software . .40 Other Assay formats. 54 Reaction Components: Principles of Quantification . .57 – MgCl2 concentration . 40 Melting Curve Analysis . .57 – dNTP concentration. 41 1.9. References . 59 – Choice of Polymerase . 41 – Hot Start Techniques. .42 – Other factors . .42 2. Working with RNA . 59 Cycling Profile for standard PCR . .43 2.1. Precautions for handling RNA . 60 Standard Pipetting Scheme . .44 2.2. Commonly used formulas . 63 Standard PCR Temperature Profile . .45 Examples. 64 2.3. Isolating and purifying RNA . 65 RNA content in various cells and tissues . .67 III 2.4. Analyzing RNA. 68 3. Working with Protein. 81 Concentration via OD Measurement . .68 3.1. Precautions for handling Proteins . .82 Purity via OD Measurement . .68 Inhibition of protease activity . .83 Size estimation of RNA fragments . .69 Stabilization . 86 2.5. RT-PCR . .70 3.2. Conversions from Nucleic Acids to Proteins . 88 Reaction Components: Information transfer . 88 – Template . .70 The Genetic Code . 89 – Choice of reverse transcription primers . 71 Characteristics of Amino Acids . 90 – Design of reverse transcription primers . .72 Codon Usage . 91 – Template Design . ..
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