A problem with plasmid DNA extraction - (Nov/11/2008 )
I wanna consult every master-hands here with a problem I had just met in previous days. Transformed bacteria grows well in antibiotic medium, but the plasmid can not be extracted. 
I picked up my only one transformed bacterium on the Amp(+) LB plate after 16-20 cultivation(A little bit longer? ) Then , I mix it into my liquid LB medium (It propagated very well) and next day I used OMEGA Plasmid Minipreparation to extract the plasmid. The key question is here, when I add Solution III into the tube, I found out that the protein precipitation was not immediately and adequately formed like normal circumstances(The underside solution in the tube looks also not so clear). In the end, there is no band appeared on the agarose gel electrophoresis.
I suppose that it was not really my monocloned transformed bacteria, but a miscellaneous one. What are your ideas? Could anyone here tell me the reason why Solution III could not normally form a protein precipitation?
Hope for your help.
Try grow the culture in Amp (+) liquid LB media, and then collect the culture for plasmid extraction using kits.
I picked up my only one transformed bacterium on the Amp(+) LB plate after 16-20 cultivation(A little bit longer? ) Then , I mix it into my liquid LB medium (It propagated very well) and next day I used OMEGA Plasmid Minipreparation to extract the plasmid. The key question is here, when I add Solution III into the tube, I found out that the protein precipitation was not immediately and adequately formed like normal circumstances(The underside solution in the tube looks also not so clear). In the end, there is no band appeared on the agarose gel electrophoresis.
I suppose that it was not really my monocloned transformed bacteria, but a miscellaneous one. What are your ideas? Could anyone here tell me the reason why Solution III could not normally form a protein precipitation?
Hope for your help.
My friend, I faced the same situation before and I think your suspicion is right. In my case, I conformed latter that it's contamination and it's Gr+ bacteria, which has the thick peptidoglycan layer and you can not lyse the cell with the normal lysis buffer used for Gr- bacteria. The neutralization buffer will precipitate the proteins released from the cell after lysis, but if the cell is tolerant to the lysis buffer, no protein released, so no white cluster of precipitation appeared. Normally, the Gr+ bacteria have low resistance to Amp, I think, because the penetration of Amp is better than Penicillin, which these bacteria frequently resist, but sometime, the condition of laboratory created some mutants show the multiple tolerances. The best way is transform again and if your vector has more than 1 selection marker, use them all, instead of only Amp.
Thank you, friend from Vietnam. Your analysis is pretty correct, multiple antibiotic tolerance is really a big headache in the lab. What's worse, the efficiency of transformation in my ligated product is rather low. Sometimes there are only one or two colonies formed on the LB plate. I will follow you advice of trying double selection marker. Thx again for your help
I picked up my only one transformed bacterium on the Amp(+) LB plate after 16-20 cultivation(A little bit longer? ) Then , I mix it into my liquid LB medium (It propagated very well) and next day I used OMEGA Plasmid Minipreparation to extract the plasmid. The key question is here, when I add Solution III into the tube, I found out that the protein precipitation was not immediately and adequately formed like normal circumstances(The underside solution in the tube looks also not so clear). In the end, there is no band appeared on the agarose gel electrophoresis.
I suppose that it was not really my monocloned transformed bacteria, but a miscellaneous one. What are your ideas? Could anyone here tell me the reason why Solution III could not normally form a protein precipitation?
Hope for your help.
Dear Wolfgang, your post is really interesting....I think I have the same problem
Yesterday I posted a problem I guess I have with the ligase....but in truth, a couple of time, I found 2-3 colonies on my dishes, after the transformation. Then I picked them up, put them in LB overnight. The day after, extraction, controls cut and agarose gel and....nothing, like you! So I thought the problem was the ligase, I thought this bacteria were just contaminating AMP-resisting bacteria...but this post gives new light to my problem!
One thing I noticed, was that this time the bacteria grew differently in the 15ml tube. They were kind of attached on the bottom of the tube, much more sticked than usual. Did you see something similar?
I don't have double resistance plasmids. I used DH5alpha E.Coli strain, do you think that shift to TOP TEN strain could change something?
Hi Niki, I don't think it is the problem of ligase(Toyobo Ligation High Kit), neither is the DH5a. Because one of my classmate just used the same DH5a to transform successfully. I suppose the key problem is more or less the concentration of template DNA. To common sense, sticky end self-ligation shall be high! I hold that I did not adjust the template concentration well.
The next day I did not find any apparent difference with the LB culture, I mean, it seems like normal, but abnormal protein precipitation occurred as I added into Solution III. 
The next day I did not find any apparent difference with the LB culture, I mean, it seems like normal, but abnormal protein precipitation occurred as I added into Solution III.
@wolfgang: You can recognize the problem sooner in this case, I guess after adding the SolII, the mixture doesn't come to be clear, even you incubate for the longer time, just because the cell isn't lysed, so the turbidity unchange.
Why don't you try the phosphatase like CIP or SAP to exclude the phosphat group from your vector, this could prevent the self-ligation of the vector. Moreover, consider the molar ratio of vector to inserted DNA, change it depend on the size differences between them (eg. molar ratio of vector to fragment = 1:3 or 1:5 or even higher).
One more thing, I think it's better to re-prepare your competent cell becuz' I think this is the easiest way to be contaminated (with the plate preparation, it involves the high temperature of you agar, so it's more difficult for the contamination to live) and remember to prepare competent cell at the best period of growth (i use the electroporation, so i collect my cell when they reach the OD600 about 0.4 or 0.9, it's very critical when you transform the ligation mixture).
@NikiITALY: I think the phenomenon you observed is not irregular, sometime I saw the same thing, even it's my pure cell. The most difficult case is the contamination colony has the same appearance as your bacteria but as my experience, something could be different. You can compare the smell from you cultured media (in the case of LB, my contamination smells terribly
) and take notice to the growth of your inoculated cell. Normally, I think the natural borne contamination shows the high rate of growth, compared with the lab strains.My "proposed mechanism" of the cluster in the bottom of inoculated media is: the E. coli cell is quite sticky, if the rate of shaking in the incubator is not high enough or other labmates opened the incubator more frequently than often, some of the cells would come to the bottom, attach to the tube wall and the others would stick to them, and so on... If you hand-shake the tube, it should come back to the normal appearance if you using E. coli, I guess. In the case of Pseudomonas, like I saw, the cluster was very tough and I can't resuspend them.