I do serial dilution of phage display to 10e10, then infect TG1 at 37℃ for 30min, the plate the mixture on 2*YTA, incubate at 37℃ overnight . However, I do not find any colony on plate .
dose anayone know what the exact reason ?
Do you know the phage titer in your initial sample? Perhaps viral particles are too diluted and you are doing too high dilutions. Did you also plate bacteria infected with more concentrated phages? You could try to use a control phage sample with known titer to determine whether the problem is related to your phage sample or to the infection procedure
How did you prepare TG1? Are them in exponential phase of growth? Did you grow them at 37 ºC until infection?
Ideally you should always grow TG1 cells on minimal media and pick colonies from there before growing in liquid media to keep them infectable. I have ommitted this step for years without any problem (I use colonies from 2xYT plates), but a colleague of mine who did the same got TG1 cells that were able to grow, but no longer infectable. Are you using minimal media at some point?
TG1 have been used for years in my lab with excellent results, but if you have problems, you could switch to other E.coli strain (XL-1Blue, DH5alpha, Top10F'...) for phage experiments.
Gertrudis Rojas
Hi Gertrudis, Thanks for you advice.
I am not sure the exact phage titer, so I do lilution to measure the titer. I diluted it to 10e10, 10e12, but only several colony grow on 2×YTplate.
For TG1, streaked plate was store at 4-8 ℃ after incubation at 37℃ overnight， then I pick a single colony and inoculate in 5ml 2× YT medium at 37℃ to prepare TG1 for infection。
So would you please tell me how you grow TG1 and the store plate ? Would you pick the colony after storing at 4 degree for infection?
Gertrudis Rojas is correct, the problem may be that you have diluted too much. There are very few phage that will give titers that high. Try a dilution series from 10e4 to 10e9 and see what you get. I presume you meant to type that you are looking for plaques on a lawn (and not for colonies?), or are you selecting for a phage marker? What phage are you using?
As I told you, ideally TG1 colonies before infection should be grown on a minimal media plate instead of 2XYT. Then you can store the colonies at 4 degrees without any problem. I always use 2XYT for that with good results but its not the textbook procedure.
On the other hand, if you dilute the phages a lot and a few colonies are observed, perhaps everything is normal, and you are just seeing the expected number of colonies. Or the numbers are definitely lower than expected?
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