Hi all,
I am trying to make a construct by Gibson Cloning (NEB Kit). I have assembled 4 fragments and the total size is 6.5 kb. These fragments contain some intronic sequences from zebrafish and also GFP. I have followed exactly the same protocol stated on the NEB website to assemble these fragments. I got 10-12 colonies. The competent cells are also from NEB (C3019, highly efficient competent). I inoculated these colonies in 3 ml ampicilin medium for miniprep but I got very small, hardly any visible pellet. I used home-made reagents. Then I loaded 4.5 ul on the gel (1%). To my surprise, there was a band of thin line width otherwise we normally get a blob on the gel if I load 4 ul. This band was out of the ladder's range. I assume that this could be nicked circular form of the plasmid. I also see one band at 1.25 kb. I am not sure origin of this band or it's a highly supercoiled form of the plasmid. Since I wanted to carry out RE analysis to confirm, I tried to perform midiprep. I inoculated a single colony in 50 ml ampicilin medium and shook for 12-14 hrs. That's how I normally do. I used QIAGEN midiprep kit. Again, I could hardly see any pellet. When I measured the conc., it turned out to be only 17 ng. Still, I loaded some 15 ul out of 20ul on the gel, I do see these two bands but the problem is the yield is too low. I can't use it for any further work. I am re-transforming the remaining plasmid into home-made DH5alpha cells if these commercial cells are having any problem of retaining plasmids in the cells. Another approach I learnt from the research gate is to quadruple the amount of antibiotic. I will also do that. If anyone has any suggestion or faced the same problem, let me know.
The 10beta cells need extra time. They are very good for large plasmids and high transformation efficiency. But you pay for that with growth rate and yield. Another strain, like 5-alpha would certainly do better.
I'm guessing you'll probably be fine if you just give them more time. It's a good suggestion to monitor the OD. Also just look at it by eye. If it doesn't look as cloudy as normal, then that explains your small pellet.
1) Last year we have realized that there appeared a yield problem with Qiagen columns unfortunately. Try Zymo mini.
2) If you use a Low copy plasmid, make a standard alkaline lysis (Sambrook) ommit RNAse treatment, precipitate DNA with isopropanol. Then dissolve the big precipitate in water with RNase, and use the column as described in the protocol. Yields are fantastic.
Dear All,
Thanks a lot. I figured out there is some problem with the kit, itself. But, your suggestions are quite valuable, maybe I can use them sometime later. :)
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