SEA-PHAGES Lysate Archiving Protocol

Transcription

1 SEA-PHAGES Lysate Archiving Protocol Objective The purpose of this protocol is to communicate the new procedure for sending phage lysates to the University of Pittsburgh to be archived. A comprehensive archive of lysates for phages found through the SEA-PHAGES program and entered into PhagesDB.org is a valuable asset to the SEA-PHAGES community as well as the larger mycobacteriophage research community. The University of Pittsburgh has agreed to house this archive and distribute samples when necessary. Set forth in these instructions are the procedures needed to ensure your institution s phages are a part of this important resource. Major Changes from Previous Years 1. We will need a Material Transfer Agreement (MTA) between the University of Pittsburgh and your school. You can begin the MTA process today by completing the attached paperwork. Once you collect the phages (referred to as SAMPLES in the MTA), we will attach the phage list to the MTA to complete it. An executed MTA is required for all archived samples to be received and distributed. 2. We are using DMSO as a preservative, not glycerol. Overview There are 3 sections of this protocol. 1. Documentation Part 1: Material Transfer Agreement Part 2: Packing Slip 2. Sample Preparation 3. Shipping Instructions Materials This year, all materials are included in the archive materials box that we will send. These include: Bar-coded screw-capped Microcentrifuge tubes (2/lysate) These tubes have unique barcodes for tracking in our inventory. The tube(s) for your school inventory is not provided. Beads (Fisher Scientific 3mm glass beads solid Catalog # A List price: $17.28). You will need to autoclave the beads to sterilize. You will want to autoclave the beads in a vessel that you can pour the beads from easily. DMSO Dymethyl Sulfoxide (Sigma Aldrich 25 ml bottle Catalog # D ml List price: $35.90) Ready to use. Shipping packaging (shipping box, shipping label, tube rack, cold packs). The box we are using to ship will also be the one you use to return the archive samples. Freeze the cold packs upon receipt so they are ready for the return trip.

2 Phage List Print-out. Please complete the contact information and required information. This information the packing slip - was generated at phagesdb.org. If it is incomplete or incorrect, please correct the information at phagesdb.org and request a new packing list by sending an to debbie at djs@pitt.edu. Phage Lysate, preferably with a titer of at least 10 9 (Provided by you!) We ask that you make 3 samples for archiving purposes: one to be kept at your home institution, and two to be sent to Dr. Graham F. Hatfull at the University of Pittsburgh (Pitt). The following protocol is to be used in preparing your phage samples that you will send to Pitt. You will receive your box in early February. Procedure Documentation, Part 1: Materials Transfer Agreement In order to accept and distribute the archived phages, the University of Pittsburgh requires a Material Transfer Agreement (MTA) for the phages that you will send. It would be great if you would process this paperwork immediately! The MTA consists of two parts, the signatures required for execution of the document and the list of SAMPLES, i.e. phages. Included in this protocol sheet is the required MTA. It requires two signatures from your institution, the instructor (only one instructor signature is necessary) and an authorized individual for your institution. The authorized individual will vary amongst institutions, so we ask that you identify that person for your institution. Some institutions may require that a different form be used. A second part of the MTA is the list of the phages (which we will refer to as the Packing Slip). This part will be generated in February and will be sent with the Archive box. Please return this list when you send us the phages. The completed MTA document consists of both parts. When complete we will make a signed copy of this form available to you. Documentation, Part 2: Packing Slip All data is to be entered at phagesdb.org prior to the generation of the packing slip. Packing slips will be generated on February 1, Please make sure that ALL of your phage entry data for this school year is entered on phagesdb.org by January 31, When you receive your archive materials box, review this sheet and make sure all information about each phage is accurate and complete. Revise all discrepancies. Revise any information at phagesdb.org and request a new packing slip. Include this printout (your packing slip ) in your archive materials box that you send back to Pitt. See SEA- PHAGES Packing Slip example sheet attached to this document. Sample Prep (for each phage you are submitting) We ask that you prepare a sample for your own archive as you prepare the phagesdb samples. Add at least one more tube (not provided) for your archive set. The following directions refer to the tubes shown in the photo below (A K).

3 1. Labeling 2 bar-coded Microcentrifuge tubes (A) with the Phage Name. (C) Ensure that the phage name is legible. 2. Fill each tube (using aseptic technique) with sterile beads to ~ 1.5 cm from the top of the tube. (D) This will keep the final mixture away from the tube threads (B). 3. Prepare the Lysate and DMSO Mixture for all of your tubes. The DMSO is provided ready-to-use. You want a final concentration of 6% DMSO. Final volume of Mixture needed is at least 3 ml. To prepare a 5ml final volume (because the pipetting is a bit easier), add 300 ul of DMSO to 4700 ul of Your Phage Lysate. Vortex. The mixture is now ready to dispense in the 3 tubes. 4. Pipette the mixture into each of the labeled tubes so that the beads are just covered. (E&F) Examples of over and under filling are included to help identify what NOT to do! (G, H, I, J) 5. Take care to close the tube properly. 6. Place the bar-coded tubes in the box provided in the same order as that designated on your packing list. That will facilitate the handling of these tubes upon their arrival at Pitt. Refrigerate until you are ready to ship. 7. Place your own archive tubes in an appropriate rack and freeze.

4 Shipping Instructions Once you have the samples prepared and necessary paperwork completed, you are ready to ship. A UPS shipping label has been provided. Using the box you received with the archive materials, pack the box of samples (surrounded by frozen cold packs) and the checked packing list. Send this box to: Hatfull Lab Mycobacteriophage Archive Samples 365 Crawford Hall Pittsburgh, PA (412)

5 University of Pittsburgh Date: Material Transfer Agreement Provider: Scientist(s): Institution: Mailing Address: Recipient: University of Pittsburgh Office of Research 123 University Place, Lower Lobby Pittsburgh, PA Recipient Scientist: Dr. Graham F. Hatfull 376 Crawford Hall Pittsburgh, PA Re: Receipt and Distribution of Archived Phages Dear : In accordance with SEA- PHAGES and phagesdb.org specifications, you agree to send an aliquot of the phage(s) that you have found and added to the database(s) dedicated to the SEA- PHAGES project ( MATERIALS ). The University of Pittsburgh ( Pittsburgh ), acting as the archive center, will maintain the SAMPLES and distribute them to SEA- PHAGES members and non- members with notification to you and your institution by e- mail at the addresses listed below, or as may be updated from time to time, and in accordance with the terms of this letter agreement ( Agreement ). The MATERIALS are provided and accepted subject to the following terms and conditions, binding on both Provider and Pittsburgh: 1. The MATERIALS, and all unmodified derivatives and progeny thereof, remain the property of Provider. 2. MATERIALS, as well as unmodified derivatives, progeny, and modifications thereof, may be distributed to members of the SEA- PHAGES community by Pittsburgh under a material transfer agreement at least as protective as this Agreement. The distribution of samples, including MATERIALS, will be at the discretion of Dr. Graham Hatfull, as lead scientist of the SEA- PHAGES program. 3. Neither the MATERIALS nor any unmodified derivatives or progeny thereof shall be used: (a) in any product, (b) for the purpose of producing any product, or (c) for providing any service in which a product or service is sold or otherwise made commercially available. I # XXXXXXX

6 4. The MATERIALS: (a) are to be used solely for teaching and academic research purposes; (b) will not be used in human subjects, in clinical trials, or for diagnostic purposes involving human subjects without the written consent of Provider; (c) will be used in compliance with all applicable statutes and regulations, including regulations related to the disposal of hazardous materials. 5. The MATERIALS shall be used with prudence and appropriate caution in any experimental work since not all of their characteristics are known. THE MATERIALS ARE PROVIDED WITHOUT WARRANTY OF MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE, OR ANY OTHER WARRANTY, EXPRESS OR IMPLIED. Except to the extent prohibited by law, Pittsburgh agrees to release Provider from any liability in connection with Pittsburgh s acquisition, use, storage or disposal of the MATERIALS. 6. Either Pittsburgh or Provider may terminate this Agreement by giving sixty (60) days written notice to the other. If MATERIALS are removed from the Archive Inventory, the related data is subject to removal from the database. If you should leave Provider s employment, Pittsburgh shall retain the MATERIALS on the terms herein unless Provider terminates this Agreement. 7. MATERIALS are provided at no cost. If these terms and conditions are acceptable to you and your institution, please sign this Agreement, and also have it signed by an authorized official who can bind Provider to these terms, in the places provided below. Very truly yours, I agree to the above terms and conditions: Kelly Downing Associate Director, Office of Research Instructor Date Printed Name: Title: address: Authorized Individual for the Institution Date Printed Name: Title: address: I # XXXXXXX