Phagehunting Protocols
The following is a list of the protocols available for phagehunting for Mycobactriophages. The first selection is our current manual of protocols. It is in serious need of revision. The remaining protocols will be better updates, but to date have not been validated. As phagehunters use the protocols, we want to encourage you to add comments to the blog to help others with any successes or problems that you encounter. Happy Phagehunting!
Ordering Info - Check this document for items that are typically used to phagehunt.
Media & Reagent Preparation - You can find how we makeour materials and grow our bacteria here.
Mycobacteriophage Isolation, Purification, Amplification - This document will get you to the 30 plate preparation for amplification of your phage. You can leave questions or comments about these protocols as a comment to this post.
Mycobacteriophage Harvest through Ligations - This document will get you from collecting the supernatant of a flooded 30-plate phage infection to ligation mixtures that are ready to be cleaned up for sequencing. You can leave questions or comments about these protocols as a comment to this post.
DNA Restriction Enzyme Digests - Here is our current protocol for our digest. Look for virtual digests in Phagebook. Coming soon!
Sequencing - New sequencing techniques are developing ata very fast rate. Until recently, we have used our in house ABI 3730 Seqeuncer and Genome Center staff to guide us through the seqeuncing process.
Sanger Method of sequencing uses dideoxynucleotides in a chain termination method to determine the order of the ATCGS. Click here for more information about the method.
Pyrosequencing method is a sequencing by synthesis method. To understand this process better, click here.
Finding the Ends of the Genomes - Presently we are using both 454 and Sanger sequencing to finish genomes. Here is tutorial for making end determinations. This presentation identifies the types of ends to the phage genomes and what data supports the determinations.
Annotation Guide (with DNAMaster version 5.0.2) - This document (last updated June 1, 2009) will guide you through the steps to gather and process the information you may use to call genes. DNAMaster is undergoing daily revision. Since the last protocol was written, the main revision now allows BOTH Glimmer and GenMark to be brought into DNAMaster for simultaneous evaluation.
You can leave questions or comments about these protocols as a comment to this post.
Created: 2008-01-16 12:46:00
Last modified: 2009-06-01 08:41:19