I'm currently working on my fourth try of transformation. Every time the plates have A LOT of tiny colonies (photo attached). Every once in a while there is a larger colony, but when I screen them, they don't contain the recombinant plasmid. The negative control has colonies also, but the plate doesn't have the tiny colonies covering it like the other plates.
I've redone the digestion and still get the same results. I checked to make sure the plates contain ampicillin by transforming bacteria with a plasmid resistant to kenomycin. No bacteria grew when I did this. I've tried troubleshooting, but there are a lot of things I can change, and I'm not sure where to start. Is the ampicillin concentration in the plates not high enough (I've been using 500 uL for 500 mL of agar), am I using too much of my ligation reaction (I've been using all 10 uL), am I plating too much (I've been plating about 350 uL)? Has anyone had this issue before?
Also, when you screen your colonies, what else do you run on the gel? I vaguely remember doing cloning in another lab and when we screened our colonies we ran the uncut vector and the cut vector, but I've only been running the negative and positive controls.
Looking at the picture it doesn't sound as if anything is wrong. You are only plating too many cells, and due to competition for nutrients, etc. the colonies are small. Try plating 1/10 of what you are using.
The only red flag is the growth in your negative control plates. Does it look like the growth in the plate displayed above? When the ampicillin is degraded or at lower concentrations than expected, both the negative control plates and the ligations either get you a lawn or lots of satellite colonies around resistant colonies, and that is not what it looks like from here.
Hope it helps!
Alejandro Martin No the negative control plate does not like the one above. There are fewer colonies, and they are larger.
There might be a whiff of AmpR contaminants among your competent cells, then. The antibiotic selection looks like it's working; I like using 100 ug/mL Amp rather than the standard 50 ug/mL, especially when working with high copy number vectors, but still the plate you showed looks OK.
Just plate 50 uL of the remainder of your transformation mix if you stored it at 4*C, and do minipreps from the most abundant colonies; you should be fine.
I agree with Alejandro Martin that it looks like you just plated too much of your transformation culture. I often plate 20ul on one plate and 200ul on a second plate so that one of them has the right number of colonies.
Did you overcome this problem@ Morgan Sedorovitz . How you got large size colony? Is the small size colony has insertion means transformation?
If you want to make sure there are enough viable colonies from that 350ul, try spinning down that larger amount, removing supernatant, resuspend bacteria in 100-200ul and spreading only that 100-200ul/plate. I've found this to be more successful than when I've spread 300ul+ in the past. Best of luck!
For some times, if the thickness of one plate is larger than another, it would be more likely to cause much larger colonies in thicker one. This is because your cell were obtaining more nutritions. Fewer cell competing for LB source could be another explanation too.
I am having the exact same problem, done the same controls and still trying to guess what's wrong
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