Day 21 thoughts.  Phagefinders. 

Planned:  Regarding DNA preparation, Blake had good results using 1 mL 10^9 PFU High Titer phage preparations.  His DNA was suspended into 100 uL of water at the end of the procedure, and he would use 5 uL of this DNA for restriction digestion.  We will take the same approach.  Each 20 uL cut will contain:

2 uL buffer,

1 uL BSA,

1 uL Enzyme,

5 uL DNA.

The digestion will occur at 37°C for 2-3 hours, and will then immediately be loaded onto a gel.  During the 2-3 hour gap, we will run 5 uL of each DNA sample in a different gel to determine whether there is any DNA or not, running the DNA for 5 min at 100 V.  No DNA? The student should have time to prepare new DNA today so that digestions can be done tomorrow with that DNA...the reason for having 2 digestion days. 

Did:  Students prepared F2 high titer for DNA preps.  Photography and further description of plaque morphology ensued.  All students have at least one F2 phage today, 2 students only have a single F2 phage and have other phages at F1.  The students that have only 1 F2 phage will be given D29 phage to also cut with restriction enzymes tomorrow.  Today, we will outline the procedures that we will do tomorrow and the day after.  Blake, our return student, made some of the solutions for DNA restriction enzyme digestion. 

We are also trying to find Rhodococcus phage, which may or may not exist.  Our collaborator from Harvard has shipped us a sample of this equine bacteria and we will begin phage-finding for this bacteria in the periphery.  Why? So our collaborator can develop a microbial genetics system for this other bacteria. 

We used photoshop for awhile today, manipulating some of the plaque images in attempt to learn if we could visualize them better, or actually, more artistically for communication/display reasons.  Lazlo is comparing the NCBI genome sequences of all phage

today's materials:   

Pointers:  some of our students