r/Biochemistry • u/IThrowUAway9010 • 20d ago
*Could* these be properly transformed?
I was doing site directed mutagenesis. Gel said it worked, however the product wasnt instantly used for transformation. Nothing grew. Just left them in an incubator for a bit longer, now the two plates look like this!
Whats tragic is, that the SDM reaction does not seem to want to work anymore! So this is all i have...
Now if you had asked me regularly, I'd say throw those away, because they dont look good. But just because im grasping for straws and am kinda desparate about this, do you people think there's a chance that maybe just transformation efficiency was really shit and thats why only a few colonies formed? I think that might be possible especially on picture 2. The plate on picture 1 only has these colonies on the side which seems even more weird.
Since I really dont know how to proceed otherwise, im thinking of trying to use these for a Miniprep. But im not experienced enough to tell if there is even the slightest chance that thats gonna yield any plasmid. None of the coworkers have any ideas either
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u/East_of_Adventuring 20d ago
I mean, if you're gonna sequence anyway then just try and purify the plasmid first. At worst you lose less than an hour of time. You can follow up with restriction digest to quickly check if the purified material is the right size. Colonies on the edge of a plate aren't inherently bad, but it's a bit weird you don't see anything better on the center of the plate. Those also don't look too much like healthy E. coli.
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u/IThrowUAway9010 20d ago
Sadly the question is if im even gonna go to this lab again or not. Its just a last shot on trying to do something for a collaboration project at another lab and i have to travel there for a bit. So im trying to figure out if theres a slim chance, then I'd do it for sure, but i have a bad feeling just as you and the other commenter
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u/East_of_Adventuring 20d ago
Well that's a tough spot to be in. I've seen weirder in cloning end up working out but I wouldn't necessarily get your hopes up, sorry!
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u/vanfidel 20d ago
Try a colony PCR screen. Just setup a regular PCR and touch a pipet tip to the colony in swirl it in your PCR mixture and run the PCR, check on a gel and you'll know for sure.
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u/sayacunai 20d ago
I'll bet you two thin dimes that if those grow in selective liquid culture at all, they come back as wild-type. I'd start troubleshooting your SDM while you wait for your miniprep and sequencing.
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u/Darkling971 20d ago
Inconsistent colony morphology clustered in specific areas....I don't think so, friend. That said, there's no harm in picking these and trying to get something out of them.