For the past two weeks Jackie, Scott and I have been trying to find the lysin gene in Amigo but we still have inconclusive results. We thought that due to Amigo being highly lytic that it would not be as difficult as it has been. Using phamerator, we have been going through found genes for ones with the function of a lysin and then looking them up in the NCBI database. We compared each one to amigo but so far the best match had an E Value of .056. Hopefully today we will find something!!
On Wednesday in lab, my group and the other group that annotated the same genes that we did debated over certain genes and whether or not they would be the longest ORF or the second longest. My group had picked the longest due to the fact that we wanted to shorten the gap however after discussion with the other group we conceded that the second ORF was a better fit, receiving a closer blast match. I really enjoyed that class period because I liked seeing what their thought process was when they were annotating the same genes as we were and how we thought similarly and how we differed.
Today in lab I found out that all ten plates of my ten plate test and my spot test were all contaminated! I think that it is the Top Agar because my negative was also contaminated. It looked like I had a webbed plate but I didn’t want to flood a plate that was contaminated so I decided to redo both tests. I made new Top Agar and made sure to be extra careful about using the aseptic technique. I am doing two tests at once (the 10 plate test and the spot test of my new lysate to find the titer) to make sure that I don’t have to back track. Hopefully on Wednesday I will get good results and be able to move forward!!
On Wednesday in lab I spent the entire class coming up with the calculations for what concentration of phages I would need to make my webbed plate. After many attempts and crossed out numbers, I came up with 14 as my ideal number! According to my calculations I need 14 micro-liters of the 10(6) dilution to make my webbed plate. I am also plating 28 and 56 micro-liters of 10(6) and 7 and 3.5 micro-liters of 10(7) incase my math was slightly off. I am hoping, praying, and crossing my fingers that I calculated correctly and I will have a perfectly webbed plate on Wednesday! Wish me luck! 🙂
I have finished all three rounds of purification and yesterday I flooded my 10(-1) plate. My phages are extremely small but there are hundreds on each plate. The top agar on my 10(-3) plate slide so I had to count my 10(-2) plate instead and there were 795 phages! By the time I had finished counting I was seeing little dots everywhere! I have been trying to think of names for my phage so I would have some ideas when the time comes but it’s really hard, I think I may just pick a name from a hat at random because I can’t decide!