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(無題) 削除/引用 No.903-8 - 2008/04/04 (金) 19:30:56 - Postgrad I linearized the plasmid and did agarose electrophoresis again. It looks like that the unknown band was linear plasmid as there was only one band (and very pale band, probably nicked) this time. Thank you very much again for your advices.

(無題) 削除/引用 No.903-7 - 2008/04/03 (木) 13:23:07 - おお Be careful about nicks. Nicks mean breaks. Transcription stops at a nick, replication as well if it is not repaired. If you get nicks in both strands, you might get linear plasmid after it gets warm. I think 10% nicked DNA in your plasmid would be acceptable. When I get much nicked DNA, I would start from E. coli culture. You might be able to purify supercoiled DNA from agarose gel, but I have not tried yet. Ultracentrifugation in CsClis the well established method by which you can separate supercoiled and nicked plasmid. However, few people take this method for purification now, even if it used to be popular.

(無題) 削除/引用 No.903-6 - 2008/04/02 (水) 20:06:52 - A >Just in case if it was nicked plasmid, can I used the plasmid solution for transfection or should I extract supercoiled plasmid from gel to remove nicked one? At least by my experience, I've never seen any plasmid solution that only contains super-coiled plasmid and never heard any trouble by it from my colleagues. So I would say that you can just move on. If you have huge amount of plasmid and enough time, it would be one choice to be sure. Also I would not do it if I were you.

(無題) 削除/引用 No.903-5 - 2008/04/02 (水) 18:15:52 - Postgrad Thank you very much for your replies. Mr. OO, indeed I did not linearized the plasmid. I compared the bands for the samples (missense mutant) with the one for plasmid (wt) given from the other lab and I thought it is enough to check molecular weight. I suppose I was wrong. I will linearlize the plasmid and see if the unknown band is nicked or linearized plasmid. Just in case if it was nicked plasmid, can I used the plasmid solution for transfection or should I extract supercoiled plasmid from gel to remove nicked one?

(無題) 削除/引用 No.903-4 - 2008/04/02 (水) 15:22:16 - おお By the way, when you pick up a single clone, it has a single plasmid mostly. So it is unlikely that all clones you pick up are contaminated, if nothing is wrong. Best,

(無題) 削除/引用 No.903-3 - 2008/04/02 (水) 15:01:19 - おお Hi, I would just like to make sure that you linearized your plasmid with some restriction enzymes. When you electrophoresis a plasmid which is not linearized, potentially you get 3 types of bands, supercoiled (intact circular), nicked and linearized forms. Usually, supercoiled forms migrate faster than the linearized form. However, you can not predict where they show up. Nicked ones migrate very slowly. Even you have 3kbps plasmid, they would appear at a size of more than 20kbps. Under the proper culture and purification condition, you can get an intact circular plasmid (more than 90%). In this case, electrophoresis shows a major band which is smaller than you expected and very week signals at the proper size and/or very large size. Even after plasmid is linearized, if the enzyme is dead, you would get multiple bands consisting of linearized and supercoiled forms. So you have to be careful about enzymes you use, as well. Check these and if you are still stuck there, just ask again with some more ditails. Good luck!

(無題) 削除/引用 No.903-2 - 2008/04/01 (火) 19:26:14 - カイアジェン 小さい方が閉環状分子で大きい方が開環状分子では？

2 bands appear after midiprep -contamination? 削除/引用 No.903-1 - 2008/04/01 (火) 18:21:55 - Postgrad Hello, I got a problem with plasmid purification after transformation and would appreciate it if you could help me. (I am sorry writing in English but somehow I cannot write in Japanese at this website.) I ligated pcDNA3.1 vector and the insert (8.1kb), of which product was used for transformtaion (ONE SHOT TOP10, invitrogen). I got 20-30 colonies on the Ampicillin plate and picked up 4 colonies. Then I incubated each at 37 degree C overnight and extracted plasmid using Midi prep (Qiagen). Simple enough. However, when I did agarose electrophoresis, 2 bands appeared for all samples, one with expected plasmid size (vector+insert, large band) and one with larger (smaller band). I am sure I picked up single colonies thus extracted plasmid should have been derived from only one colony = one plasmid. What should I make of this larger plasmid? Is it possible that the E.coli got 2 different plasmids? Or is it just a contamination?

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