I am doing recombineering and would like to swap out GFP for mCherry in my vector. I can proceed with the GalK selection scheme but I tried it once and didn't work so I am trying to think of alternatives. I noticed a T7 promoter with a lacO upstream of GFP so I am wondering if I can plate my recombinations on plates with IPTG and look for red fluorescence over green. Has anyone done something like this? Are you able to see fluorescence with the right scope?
Thanks a lot!
I haven't done this myself, but I believe it is doable. LB however gives really high background fluorescence so you might want to change your plate type to something with better optics. I have noticed that most of my constructs that are overproducing mCherry give my cells a pretty strong pink/red tint to them. If I am freezing a stock they are distinctly red compared to other frozen stocks without mCherry. You might be able to observe them without fluorescence depending on how much protein you are producing.
G'luck!
thanks Cara! Glad to know. Always happy to get help from a fellow deisian!
So long as you have the right imaging set up to appropriately visualize, then you shouldn't have a problem assuming reasonable expression. i've identified GFP expression that way a number of times.
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