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The Halohandbook © Protocols for haloarchaeal genetics Don’t Panic. This document has a wide range of methods for haloarchaea and their viruses. Version 7.2, March 2009 © Compiled and edited by Dr Mike Dyall-Smith, Currently I am working at the Max Planck Institute, Martinsried, Germany. e-mail: mike.dyallsmith (AT) gmail.com web: http://www.haloarchaea.com Protocols for halobacterial genetics © Ver 7.2, March 2009 page 1 A typical salt lake in southern Australia. MDS, 2004 Contributors Below are the people who have helped make this book so useful. Sources are quoted where possible. Please join this list by contributing your favourite method ! Address details of some contributors are just before the references, but google searches should get many anyway. Melissa Holmes Sen-Lin Tang Thorsten Allers Masahiro Kamekura Valery Tarasov Jerry Eichler Wan Lam Vicky Athanasopoulos Helen Camakaris Stewart Nuttall Carolyn Bath Tristan McAlpine Wayne Woods Darrow Wendoloski David Burns Peter Jablonski Consuelo Ferrer Kate Porter Juan Serrano Matthias Pfeiffer Tania Cukalac Katrina Ngui Mecky Pohlschroder Brendan Russ Josefa Antón Fernando Santos Any comments or suggestions welcome! Square haloarchaeon of Walsby (SHOW organism, strain C23), grown for the first time in 2004, 25 years after their discovery by Prof. Tony Walsby (Walsby, 1980). The name of the type strain is Haloquadratum walsbyi C23T Protocols for halobacterial genetics © Ver 7.2, March 2009 page 2 TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................................................................. 3 1.0 Introduction to halobacteria .................................................................................................................................. 6 Haloarchaeal Journals: ................................................................................................................................................10 2.0 Media Formulae and Cultivation of Haloarchaea ............................................................................................. 11 2.1 Artificial Seawater (3.6% salt) ...............................................................................................................................11 2.2 Concentrated Salt Water (SW) stock solution - 30% (w/v) .....................................................................................12 2.3 Modified Growth Medium (MGM) for Haloarchaea .............................................................................................14 2.4 CDM - Minimal Medium for Haloarchaea (Kauri et al., 1990) .............................................................................16 2.5 OTHER PUBLISHED (or unpublished!) MEDIA FORMULATIONS ....................................................................18 2.7 Solutions for Media .................................................................................................................................................33 2.8 Cultivation of Haloarchaea ...................................................................................................................................36 3.0 Transformation Methods ..................................................................................................................................... 41 3.1 Preparation of Competent Frozen Haloarchaeal cells ..........................................................................................41 3.2 Transformation Protocols (MDS Lab) ....................................................................................................................43 SUMMARY OF HALOARCHAEAL TRANSFORMATION PROTOCOL .....................................................................46 3.2.4 Freeze-Thaw transformation method ...................................................................................................................51 3.2.5 Selectable markers and plasmid replicons: ........................................................................................................52 3.3 Transformation Solutions - MDS Lab ....................................................................................................................53 3.3.7 Transformation Top Agar ....................................................................................................................................57 3.3.8 Minimal Medium (MM) Transformation-Top Agar for plating transformation mixtures ...................................58 3.4 Haloferax volcanii Transformation - Thorsten Allers Laboratory ......................................................................59 3.5 Transposon Mutagenesis of Haloarcula hispanica ...............................................................................................63 3.6 Natural Genetic Transfer (method of Tchelet and Mevarech, 1994). ....................................................................64 4.0 DNA and RNA Extraction Procedures .............................................................................................................. 65 4.1 Plasmid DNA .........................................................................................................................................................65 4.2.1 Hfx. volcanii Genomic DNA Miniprep by DNA Spooling ....................................................................................67 4.2.2 Chromosomal DNA isolation (rapid, small scale method) .................................................................................69 4.2.3 Alternative rapid chromosomal DNA isolation ...................................................................................................70 4.2.3 Haloferax volcanii DNA Prep in Agarose Plugs for PFGE ................................................................................71 4.2.4 PCR of haloarchaeal DNA. Recommended enzymes and conditions .................................................................73 4.3 RNA isolation and northern blotting .......................................................................................................................74 4.3.1 RNA isolation using a commercial kit ................................................................................................................74 4.3.2 RNA isolation using a manual method .................................................................................................................74 5.0 Haloarchaeal Viruses (Haloviruses, Halophages) ............................................................................................. 78 5.2 HF1 and HF2 - examples of lytic, head-tail haloviruses ........................................................................................79 Protocols for halobacterial genetics © Ver 7.2, March 2009 page 3 5.2.1 Haloviruses HF1 and HF2: large scale growth and purification .......................................................................80 5.3 Salterproviruses His1 and His2: examples of spindle-shaped haloviruses ............................................................83 5.3.3 His1 and His2: Large scale growth and purification ..........................................................................................83 5.4.3 Purification of halovirus SH1 ............................................................................................................................86 5.5 Titrations of Haloviruses ........................................................................................................................................86 5.5.1 Simple Method of titrating virus. ........................................................................................................................86 5.5.2 Standard plate titrations. ....................................................................................................................................87 5.6 Virus DNA Preparation .........................................................................................................................................88 5.6.1 SOLUTIONS ........................................................................................................................................................89 5.6.2 HVD. Used for dilution and storage of haloviruses ............................................................................................90 5.7 Notes on plaque assays and virus growth in halobacteria. ....................................................................................90 6.0 Whole Cell Methods ............................................................................................................................................. 91 6.1 Measuring cell growth using Absorbance (optical density) ...................................................................................91 6.2.1 Making a cell lysate of Hfx. volcanii for enzyme assays .....................................................................................92 6.2.2 Cell Free Extract (CFE) of Hbt. salinarum .........................................................................................................93 6.3 Making Gene Knock-Out Mutants of Hfx. volcanii ................................................................................................95 6.3.1 Characteristics and requirements of Hfx. volcanii mutants ................................................................................96 6.3.2 Plasmids used with Hfx. volcanii mutants ...........................................................................................................96
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