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Molecular Genetics Laboratory Procedures

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Molecular Genetics Laboratory Procedures Molecular Genetics Laboratory Procedures by Douglas Rhoads 2021 Laboratory Procedures 2021 Page: iii Table of Contents INTRODUCTION TO RULES AND PROCEDURES .................................................................. 1 Acknowledgments....................................................................................................................... 1 How to Use this Manual ............................................................................................................. 1 General suggestions .................................................................................................................... 2 Safety .......................................................................................................................................... 2 Emergency Procedures ................................................................................................................ 3 Major Spill: ............................................................................................................................. 3 Minor Spill: ............................................................................................................................. 4 Call 911 if the spill: ................................................................................................................. 5 Spills that require special procedures: .................................................................................... 5 Reportable Quantities: ............................................................................................................ 5 Laboratory Safety OSHA Laboratory Standard .......................................................................... 5 Elements of the Laboratory Standard ..................................................................................... 6 Laboratory Safety Chemical Hygiene Plan (CHP) ..................................................................... 7 Required CHP Elements ......................................................................................................... 7 Worker Training Must Include: ............................................................................................... 8 Medical Exams and Consultation. .......................................................................................... 8 Using a Micropipettor ................................................................................................................. 9 Handling Solutions.................................................................................................................... 10 Hazardous Chemicals.................................................................................................................11 Radioactivity ............................................................................................................................. 12 Handling Enzymes .................................................................................................................... 12 Handling DNA and RNA .......................................................................................................... 13 Storage .................................................................................................................................. 13 pH and Ions ........................................................................................................................... 14 Alcohol Precipitation ............................................................................................................ 14 USEFUL NUMBERS & CONVERSIONS .................................................................................. 18 STANDARD SOLUTIONS & REAGENTS ................................................................................ 21 Gel Solutions ............................................................................................................................. 21 Standard Solutions .................................................................................................................... 21 Media for E.coli ........................................................................................................................ 23 Media for Yeast ......................................................................................................................... 24 GENERAL PROCEDURES ......................................................................................................... 26 Quantification of nucleic acids ................................................................................................. 26 UV Spectrophotometry- best for quantifying oligos, single stranded RNA and DNA. ........ 26 Ethidium fluorescence- best if you have small quantities and no fluorometer ..................... 26 Hoechst fluorescence- best for small quantities of DNA...................................................... 27 Organic Extraction .................................................................................................................... 29 Disposal of waste organic solvents ........................................................................................... 32 Cleanup of Genomic DNA using Nanosep Filtration ............................................................... 32 Purification using a G-50 Centrifuge Column .......................................................................... 32 CONTENTS Page: iv Bradford method for protein quantification .............................................................................. 33 Microassay Bradford Protein Quantification. ........................................................................... 33 Quantification of protein by the Biorad DC assay .................................................................... 34 Lowry Protein Quantification ................................................................................................... 34 Quantification of radioactivity by TCA precipitation ............................................................... 34 ISOLATING EUKARYOTIC DNA ............................................................................................. 36 Preparation of Tissues or Cells ................................................................................................. 36 Blood ..................................................................................................................................... 36 Isolation of buffy coat cells- ................................................................................................. 36 Simplified preparation of DNA from blood .......................................................................... 37 Fast Isolation of DNA from blood for PCR .......................................................................... 37 New and Improved method for fast isolation from frozen avian blood in 96 well format ... 38 New and Improved method for fast isolation from fresh avian blood in 96 well format ..... 38 Cleanup of DNAs from fast isolation in 96 well format for NGS library synthesis ............. 39 Simple Isolation of DNA from Feathers for PCR analysis ................................................... 40 Simple Isolation of DNA from Semen for PCR analysis ...................................................... 40 Suspension cells or cultured cells ......................................................................................... 40 Plant Leaf Tissue ................................................................................................................... 42 Differential precipitation of Polysaccharides from DNA ..................................................... 42 Determination of cellular DNA and Protein concentrations ................................................. 43 Microwave Isolation of Total Plant or Fungal DNA for PCR/RAPD Analysis .................... 43 DNA Extraction from Feathers ............................................................................................. 43 Fungal DNA Extraction ........................................................................................................ 44 GELS: STANDARD ..................................................................................................................... 46 Ethidium Bromide (EthBr) ....................................................................................................... 46 Agarose Gels ............................................................................................................................. 46 Acrylamide DNA Gels .............................................................................................................. 47 Nondenaturing gel ................................................................................................................. 47 Denaturing TEB Acrylamide gels ......................................................................................... 48 Denaturing TEB Minigels ..................................................................................................... 48 Denaturing TBE Acrylamide gels ......................................................................................... 49 Gradient Sequencing Gels..................................................................................................... 50 Recovering Samples from Gels ................................................................................................ 51 DNA Electroelution from Gels ............................................................................................
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