Dear community,
Here is my problem: We have amplified multiple plasmids of various size with chemically competent cells from TOPO kits that were at -80°C and were possibly expired. We got no results for the PUC19 control but got results for our plasmids (confirmed by restriction enzyme digestion). We tried multiple kits and puc19 thinking that maybe we would have different results but none worked. We even PCRed the Puc19 to verify its integrity and they were good.
We proceeded with our plasmid assembly with a brand new kit of electro competent cells this time. And here again the puc19 control didn't work but we got colonies in one of the transformation plates (results to come).
We've tripled check the concentration of ampicillin as well as the protocols, try to troubleshoot multiple times with all the various solutions that we could come up with, but still we cannot get any puc19 control to work.
Maybe I should mention that the ampicillin might be old as well but then why would it work for the transformation and not for the control?
Can someone help deciphering this situation please?
Are your other plasmids also ampicillin resistance? If not then you should get a plasmid that is so that you can rule out a problem with those plates or your ampicillin concentration.
If the media is not the problem then the undoubtedly the problem is with the pUC19 control plasmid itself. The DNA may be degraded or otherwise damaged as to render it non-transformable.
Also when you say you get some results with multiple different plasmids, what sort of results are you getting? Thousands of colonies or just a few.
Other plasmids all have the ampicillin resistance and we get a good amount of colonies but not thousands.
After re checking it seems like we might have been wrong with the ampicillin concentration (its often the most obvious mistake that one might miss, we trusted ourselves too much on the conversion).
I will redo the experiment just today with the right concentration with the same transformation as yesterday (kept them at 4°) and see if that actually was the problem.
It is surprising though that the transformation worked with a higher dose of ampicillin and not the Puc 19.
Anyway, I thank you for you quick reply and will keep you updated.
I think the problem will most likely be with the plasmid DNA. If you get colonies with other ampicillin resistant plasmids then the medium and amp should not be the problem. pUC19 normally confers resistance to very high levels of amp (10-fold higher than normal) so I really doubt it is the concentration.
Well, as you predicted the Puc19 (from the brand new kit) didn't work while our transformation grew colonies on the LB plates with the right amp concentration.
Could the ampicillin be expired?
I really don't see what could lead to these results... We are not experts, it's the first time we do plasmid assembly.
If the ampicillin was not good then you would just have everything growing instead of nothing growing.
But just to be on the safe side, could you send a picture of one of your successful transformation plates, sometimes just seeing what things look like can be helpful. I presume the pUC19 plate is just entirely blank?
So turns out all the controls (assembly control and Puc19 control) developped decent amount of colonies but took just a bit more time. I suspect it might be because of the time at 4°C. T = Puc19 Controle = assembly control
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