Why the colonies bigger than usual? - (Feb/27/2005 )

Insert gene (1.3 kB) is from Topo vector, and vector is PUAST(9kb). After ligated them and did transformation, found the colones on a plate with Amp are bigger than usual and there are around 30 colones on the plate.

My question is why the colones are bigger than usual? Because I see usually the colones are small ones on plate. I used only vector PUAST plasmid without enzyme digest doing transformation, and got a lot of very small clones, but after inserted the 1.3 kb foreign gene to a 9kb pPUAST vector, and the colones on the plate look bigger. could somebody tell me why? Thanks a lot!

Did you get the plasmid expressed in the larger colonies or were they negative?

Thanks much for your reply. I am doing enzyme digesting to test if the big colones are positive or negative. But the colones which I got are really bigger than usual. Did you meet the big colones transfered before?

Hi!
I've had sometimes bigger colonies too. I've tought the plates may be moist or something, because at the same time they have seemed little bit speard. But I really would also hear a reason for this, because the phenomenon is not systematic.
Those bigger colonies were negative in my experiment. I mean they didn't contain the insert.

Cheers!

Well, we still don't know if you got any insert in your larger colonies.
The reaason I asked was that it is possible that there larger colonies are Yeast colonies.
They tend to be round and shiny. They can sometimes contaminate your plates.
If the colonies are large and flat, it may be a problem with the plate (old amp, moisture etc) and
bacterieria that do not have insert usually grow like this. However, if you actually got plamid in these large colonies- I dunno.

Yes, finally I got a positive one from these not much colonies, I did the double digest and run the gel, found the inserted band after checking a lot colonies. Do you think if it is necessary to continue to do a sequence checking?

I don't think the moist is main reason which causes big colonies, since I did the vector only transformation without digest same time, and I got a lot of smaller colonies on all of moist plate.

And the larger colonies are not Yeast colonies, and it is round, but not shiny. Thanks.