Favorite King Lab Recipes & Protocols

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NOTE FOR UNDERGRADUATES: These recipes and protocols are presented here for general information and reference purposes only. It is not intended to replace first-time hands-on instruction from an experienced lab member. IF YOU DO NOT KNOW HOW TO DO SOMETHING, ASK SOMEONE WHO DOES!

DISCLAIMER: These recipes and protocols are used by the King lab to produce and analyze Salmonella bacteriophage P22 and the tailspike protein from P22 (produced via phage infection or overexpression inE. coli ). Our methods have been refined to suit our specific systems, and therefore may not work as well when used with other systems. Good luck!

The initials used beside each recipe/protocol indicate the author:

CHP = Cameron Haase-Pettingell
ASR = Anne Robinson
SDB = Scott Betts
PLC = Patricia L. Clark
CBB = Chrisopher B. Benton
MSKC = Melissa Kosinski-Collins

Phage Protocols

• Making Plating Bacteria (CBB)
• Streaking a Phage Stock (CBB)
• Titering Phage (CHP)
• Phage Stock Preparation (CHP)
• Head Binding of Tailspikes (CHP)
• High-Titer Phage Preparation (CHP)
• High-Titer Phage Preparation (SDB)
• Making Phage Heads (CHP)
• Minimal Media for Salmonella 7155 and 7136 (CHP)
• Phage Buffer (PLC)
• CsCl Step Gradient to Purify Phage (CHP)
• LB Super Soft DF Media (CHP)

Analysis of Protein by Gel Electrophorisis

• Setting Up and Running Standard SDS Gels (CHP)
• Silver Staining Gels (CHP)
• Timeline: From phage master stock to purified protein (CBB)
• Phosphorimaging Radioactive Gels (PLC)
• Western (CHP)
• Western (ASR)
• Determining tailspike concentrations (CHP)
• Native Gel Buffers (CHP)
• SDS Gel Buffers (CHP)
• Standard-sized SDS Gels (CHP)
• SDS PAGE using criterion system (CHP)
  

Protein Preparation

• E. coli Lysis (CHP)
• Protein in Phage System (CHP)
• Tailspike Production (Older) (SDB)
• Tailspike Staggered Refolding Initiation (CHP)
• Preparation and Refolding of Tailspike Inclusion Bodies (SDB)
• Large grow pET tailspike in BL21 (CHP)

Following Proteins

• In Vivo Pulse/Chase (CBB)
• In Vivo Pulse/Chase (PLC)
• Indirect ELISA (PLC)
• Competition ELISA (PLC)
• Competition ELISA (CHP)
• Immunoprecipitation with Antibody Cross-Linked to Protein G-Agarose Beads (PLC)
• Preparing, Isolating and Characterizing Ribosomes (PLC)
• Making and Fractionating Linear Sucrose Gradients (PLC)

Molecular Biology

• Site-Directed Mutagenesiss of H gamma DC in pQE.1 (MSKC)
• Transformation of XL-1 Blue Cells (Home-grown) (MSKC)
• Transformation of M15 cells (Home-made cells) (MSKC)
• Preparing competent cells (XL-1 Blue or M15[pREP4]) (MSKC)
  

Others

• PCR Amplification of the Tailspike Gene (CHP)

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