Day 1 thoughts.  Phagefinders.

Planned.

Statement of this weeks 4 goals:  to collect new samples for phage isolation, to prepare high titer phage for Demi's project, to isolate phage from our soil samples, to learn the process of serial dilution.  Today:  to learn how to dilute phage and to prepare D29 and BXZ1 high-titer phage preparations. 

Did.  Completed an introduction to the phage, it took awhile for college student and technical help to prepare the top agar and MP solution.  Somebody took our reserved test tubes, so we used larger test tubes than normal and decided to aliquot portions of our dilutions to eppendorf tubes for incubation at 37 C (see appropriate methods and rational for what to do, below).  As a result, we plated the phage/smegmatis mixture into 1 mL top agar rather than the desirable 3, and the top-agar did not spread as well as desired across the plate.  Next time, use 3 mL top agar and make sure that the appropriate test-tubes have been rat-holed!  It was nice to leaf-thru the phage electron-micrographs from last year, to approach the cell paper and to have the returning student from last year show us his phage plates.  By the end of the day we also had MP buffer and Top Agar prepared. 

Today's materials.  2 boxes 200 uL filter tips, 2 boxes 1000 uL filter tips, 1 rack autoclaved glass 10x75mm or 6 x50mm reusable borosilicate test tubes with metal caps, 200 and 1000 uL pipettman (ideal: 1 pair pipettman for each student pair), 3 pipette pipettors (not cordless); sharpie magic markers, 1 box 5 mL sterile pipettes, writing pens for students to take notes, folders with the cell paper and blank paper for the lab-notebooks, and blank paper.  Also necessary:  37 C incubator, refrigerator.  Start up is difficult!  Needed Top Agar and MP buffer prepared for today, too, in addition to the 7H10 plates. 

Pointers:  Sterile Technique.  It is not necessary to work in the hood; phage can be plated on the counter top.  Be careful about working over the top of the media plates and solutions.  Test tube size.  It is necessary to use test tubes that are less deep than the pipetman barrel.  This allows for sterile pipetting of the diluted phage from one tube to the next.   Top-Agar plating.  Incubate 100 uL M. smegmatis with 100 uL diluted phage for 20 min @ 37°C in glass test-tubes after dilution of the phage.  For dilution of the phage, if the sample of the phage is expected to be 10^10 plaque forming units (PFU)/mL, plate 111 uL of the -4, -5, -6 and -7 (ten fold) dilutions (theoretically, this allows for 10^5, 10^4, 10^3, 10^2 PFU/plate this way.)  This protocol can be used to explain the phage dilution procedure, but is also described in part herein.   Phage Dilution.  Dilution of the phage is done by pipetting 111 uL phage stock into 1 mL MP buffer, transfering 111 uL of this stock into the next test tube containing 1 mL MP buffer, mixing by gentle flicking and transfer of 111 uL of this stock to the next 1 mL of MP buffer in a capped test tube, and so on, until the desired maximum dilution for plating is obtained.  Preparation.  For today, it is essential that 7h10 plates have been prepared, that some MP buffer has been aliquoted into one-time use portions of approximately 50 mL each, (sterile/autoclaved/filtered stock solutions should be on reserve), that the materials have been gathered (emphasis on appropriate size test tubes), and that phage has been found for students to dilute.