I've been trying to amplify KM13 helper phage using the protocol in box one from the Christ Dab library. I've been doing serial dilutions from the phage stock, infecting 200 ul TG1 bacteria at either 0.5 or 1.0 OD 30 min, at 37 C and without shaking. Then I've been mixing the infected bacteria with top agar (45 C) and pouring on warm TYE (no amp, no glucose) plates. So far I have not had success getting plaques. So, any insights please or if someone has a nice protocol please let me know.
I know this is the standard method to measure phage titers (in pfu), but alternatively you could grow the infected cells on 2xYT/kan and measure the titers in cfu. Thats simpler and works in general.
If you don't see any cfu on plate it may be because you are still too concentrate in helper phage. So your Petri Dish is empty because there is too much lysis plates to see one colony.
Hi, Gertrudis. Ho can I produce more helper phage from there? Do I need to pick one of those colonies and add more helper phage?
Yes, you can pcik a colony, grow it in liquid medium with kan and purify the phages by precipitation.
with PEG
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