What is the cause of my small E. coli colonies from transformation? - (Aug/08/2007 )
I transform DH5-alpha E. coli competent cells made from a CaCl2 protocol using a regular heat shock protocol.
After 12-24 hours, the colonies I get are extremely small, somewhat like satillite colonies. In case the Amp concentration is too high, I have remade my Amp stock and made new plates (at 50ug/ml) and will see the results tomorrow.
Has anyone else had this problem of very very small colonies?
Is your incubator really at 37º? Even if it is set for 37ºC I would read the temperature with a thermometer to be sure.
Maybe test your competent cells with another plasmid. Preferentially one that you know the concentration - try pUC18/19.
If that doesn't work maybe your competent cells are not so competent after all.
Have you tried testing those colonies with pcr? If you don't have the primers for it, you can also use those colonies to start liquid cultures to miniprep and check if the plasmid of interest is there.
And well, I doubt that this is the problem, but check if the plamid you are using is really amp resistant...
how long do you shake them after heat shock. You need to shake them atleast 40 mins before plating so that they can express the antibiotic resistance gene.
I incubate them for 40 mins- in microfuge tube. I don't shake them. I always imagined there was enough oxygen dissolved that it was okay for only 40 mins. Do I need to? Thanks scolix.
What do you know about your constructs? Is the insert coding for something toxic? Is expression possibly leaky? Both of these will slow down the rate of replication.
Good suggestion- I didn't really think of that. It could very we'll be toxic- these are mutant constructs I've never expressed in bacteria, but I don't think the expression can be leaky - it's under control of a CMV promoter.
If the problem persists, perhaps I'll post a photo.
Ambrasio- You were sooo right. The temperature of my incubator has been the source of a lot problems. Even though it was set to 37 (or so I thought) and the thermometer in the incubator said 37, it was really at 45 C! (this was determined after I checked it with three different thermometers upon your suggestion). Uh... so fustrating- but this explains why my transformations would just randomly fail sometimes.
I used a different incubator last night and got >1000 transformants all of the same size. Thanks again.
I would shake them for atleast 40 min. Try it. Make sure they shake well.
Its not only matter of enough oxygen, but proper airation.