I amplified my stock helper phage by infecting Tg1 at an OD600=0.46 for 90 minutes shaking, spinning down the bacteria, and resuspending the pellet in 2xTY and kandamycin shaking overnight. I've tried spinning the resulting solution multiple times and carefully extracting and storing the supernatant. I also tried using a .45 filter and syringe to filter the supernatant, but the filter became clogged immediately. In terms of titres, I've tried a melted top agar strategy and no plaques formed with any dilution. I've also had multiple trials of infecting Tg1 with each serial dilution and plating on kandamycin and 2xTY plates. I've only seen colonies once, and the titre was too low to continue.
Any advice would be greatly appreciated! I've been stuck on this first phase of the protocol for a long time and I'm open to any suggestions!
My protocol doesn't call for PEG precipitation for this step, but I was definitely considering it. I'm glad to hear that worked for you! I'll try that today!
so I added 50mL of my 20% PEG/NaCl, and placed my helper phage on ice for two hours. When I spun it down, I didn't see anything even remotely resembling a pellet or film.
I've read some conflicting protocols on the method of initial infection (shaking throughout vs 30 non-shaking period at the beginning). I've tried both and they didn't affect my titres.
I always infect exponentially growing TG1 cells (grown at 37 degrees) with diluted M13KO7) and plate the cells on 2xYT/kan plates. This step serves two purposes.
1. I confirm that the infection was fine. Colonies are observed in infected (and non in uninfected) bacteria plates.
2. I can start the next step using homogeneous infected cells from a single colony.
Then I inoculate one colony in 50 ml of 2xYT/kan and grow bacteria overnight at 28 degrees with shaking.
Next day I inoculate the cell suspension in 300 ml of fresh 2xYT/kan and grow bacteria overnight at 28 degrees with shaking.
After this step I collect the supernatant and start the precipitation with PEG/NaCl. After a first precipitation, I resuspend the phage pellet in 20 ml of PBS and perform a second precipitation with 4 ml of PEG/NaCl solution. While the first precipitation can be visible or not, the second precipitation must render a white precipitate (the solution looks like milk immediately after mixing the phages with PEG/NaCl). If that doesnt happen, there was no phage production and you can discard everything. If there was precipitation, I spin to obtain the phage pellet with is resuspended in 20 ml PBS, and subsequently aliquoted, and frozen.
I always determine phage titers by infecting TG1 and plating the cells on 2xYT/kan.
By the way, I infect TG1 during 30 min at 37 degrees without shaking.
Thanks everyone!
I followed the PEG precipitation protocol that you sent me, Thamsanqua! Spinning at 4 degrees C gave me much better phage pellets this time. Upon titring the final filtered supernatant, however, my plates grew practically full bacterial monolayers with no countable colonies. The phage didn't look like it diluted out, even up to the 10^14 dilution. I performed 100-fold dilutions of the phage, and infected 500 uL of exponential phage Tg1 for thirty minutes without shaking. I plated 100uL of each dilution on 2xTY plates with 12.5uL of kan. Any advice?
Did you change the tips between serial dilutions? This is quite important to avoid obverestimating the titers. Usual titers are around 10- 13 in my hands. You got too many colonies, but your phage amplification was suddessful, is that right?
Yes, the phage amplification was successful, and the precipitation went smoothly as far as I could tell. I did change tips. Maybe I'm submerging the tips too far into each dilution before the phage transfer?
You have to be very careful to avoid drops ouside of the tip. Im almost sure that the way to prepare dilutions is the reason for your excessive titers...
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating