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Dunham Lab Chemostat Manual

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Dunham Lab Chemostat Manual Dunham Lab Chemostat Manual Maitreya Dunham Last revised December 2005 This comprehensive manual covers the entire process of running a chemostat, including media recipes, chemostat setup, inoculation, data acquisition and storage, daily monitoring, harvests for DNA and RNA, and data analysis. Although I wrote it with my own yeast cultures and ATR Sixfors setup in mind, many of the procedures are general. If you have edits or additions to the manual, please email me at [email protected] Please feel free to point other people to these instructions. Also, I would appreciate the citation if you use any of this information in a publication or talk. Visit genomics.princeton.edu/dunham for the most recent updates to the protocol and for my other protocols on microarrays. Thanks to Matt Brauer, my long-time companion in the chemostat lab, for help developing these protocols and for proofing the manual. Also thanks to Frank Rosenzweig who taught me how to run my first glass-blown chemostats. Many of the protocols were influenced by his chemostat aesthetic. Edits since last version: new phosphate limitation recipe, new DNA prep, new filter-sterilization method of media preparation © Maitreya Dunham 2005 1 Contents CONTENTS ........................................................................................ 2 FIGURES ........................................................................................... 5 PLANNING THE EXPERIMENT............................................................ 6 Signup ................................................................................................6 Strains ................................................................................................6 Limitations...........................................................................................7 Dilution Rate........................................................................................9 CHEMOSTAT MEDIA .........................................................................11 Salts ................................................................................................. 11 10X salts for carbon, leucine, or uracil limitation.................................. 12 10X salts for phosphate limitation...................................................... 12 10X salts for sulfur limitation ............................................................ 12 10X salts for nitrogen limitation......................................................... 12 Metals............................................................................................... 13 1000X metals.................................................................................. 13 Vitamins............................................................................................ 14 1000X Vitamins ............................................................................... 14 Tubing and Fittings ............................................................................. 15 Carboy .............................................................................................. 15 Autoclaving the salts........................................................................... 16 Additives ........................................................................................... 18 glucose (dextrose) limitation additives ............................................... 19 phosphate limitation additives ........................................................... 19 sulfur limitation additives.................................................................. 19 nitrogen limitation additives .............................................................. 20 leucine limitation additives................................................................ 20 uracil limitation additives .................................................................. 20 Making a carboy of media.................................................................... 21 2 Attaching the carboy........................................................................... 21 Plates................................................................................................ 23 YPD................................................................................................ 23 YNB ............................................................................................... 24 Supplements for YNB ....................................................................... 25 canavanine plates ............................................................................ 26 Dropout powder............................................................................... 26 5-fluorouracil plates ......................................................................... 27 alpha-aminoadipate plates ................................................................ 27 Kanamycin (aka G418 or Geneticin) plates.......................................... 27 Assembling the vessel......................................................................... 28 Autoclaving the chemostats ................................................................. 31 SETTING UP A RUN ..........................................................................32 Polarizing the dissolved oxygen probes.................................................. 32 Setting up the vessel .......................................................................... 32 Connecting the air .............................................................................. 33 Starting the media flow ....................................................................... 33 Programming the chemostat ................................................................ 35 Profiles........................................................................................... 35 Calibrating the Dissolved Oxygen Probe................................................. 39 Calibrating the temperature probe ........................................................ 40 Inoculating the chemostats.................................................................. 40 Signup .............................................................................................. 42 Data collection ................................................................................... 43 Sample tracking ................................................................................. 45 Preparing to sample............................................................................ 45 Contamination issues .......................................................................... 46 Sampling........................................................................................... 46 Klett .............................................................................................. 46 3 Glycerol stock ................................................................................. 47 Spectrophotometer .......................................................................... 47 Sonicator........................................................................................ 47 Coulter counter ............................................................................... 48 Plating for viable counts ................................................................... 48 Plating for drug resistance ................................................................ 49 Sampling for DNA ............................................................................ 49 Sorbitol Solution.............................................................................. 49 Sampling for RNA ............................................................................ 49 Cleanup.......................................................................................... 51 Counting colonies ............................................................................ 51 Media Replacement............................................................................. 51 DATA ANALYSIS ..............................................................................54 HARVESTING ...................................................................................55 Setup for harvesting ........................................................................... 55 Harvesting......................................................................................... 56 SAMPLE PROCESSING......................................................................59 Filtrates............................................................................................. 59 Culture revival ................................................................................... 59 DNA prep .......................................................................................... 60 RNA prep........................................................................................... 60 Lysis buffer ..................................................................................... 62 RNA prep for 5 ml daily samples ........................................................ 63 RNA prep for 100 ml harvest............................................................. 64 PROBLEMS.......................................................................................65 CHEMOSTAT REFERENCES................................................................67 SUPPLIES AND SUPPLIERS ..............................................................68 ATR ............................................................................................... 68 Fisher............................................................................................
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