I did maxiprep plasmid/BAC isolation. When I did gel analyze, I was expecting for the classical two bands for the supercoiled and linear plasmids. Instead, I got real intense bands that corresponded to my cloned insert size. Nevertheless, I still see really faint bands that could represent the classical two bands for the plasmids. Does this mean my mixture has something in it that separated the insert from the vector?
Hi there - it sounds like you digested this after extraction. However, to help you, we need to know:
the size of the plasmid
the size of the insert
the amount of DNA digested
Also - what were you isolating, a plasmid or a BAC...
thanks
G
It sounds like you are analyzing uncut plasmids. Estimating sizes of supercoiled plasmids is not reliable. You are probably seeing supercoiled and nicked plasmids on the gel. Use a restriction enzyme that cuts only once, then run the gel. It is the most reliable method for size estimation.
No, your insert cannot amplify without being in the vector. It is likely that the supercoiled version of the plasmid maxi-prep may be close to the size of the original insert. The supercoiled form tends to be the predominant form of DNA in a maxi-prep. Before doing anything else, cut the plasmid with a few restriction enzymes that will give you a diagnostic pattern for your clone and see what it looks like.
My vector size is 3.1 kb. Together with the insert of, for example 1.5 kb, I should expect around 4.6 kb for the linearized plasmid and another band for the supercoiled form. However, the intense band actually corresponded to 1.5 kb, which means that my inserted DNA was cut from the vector even without me doing the restriction enzyme digestion. Is that even possible, I mean do you have similar experiences before?
No it does not mean that at all. The supercoiled form will run faster than any other plasmid form; it could be around 1.5 kb. In any case, you cannot make any conclusions without cutting the plasmid with some restriction enzymes.
While I can't predict the apparent size of the supercoiled plasmid, they run much faster in the gel that the linearized plasmids of the same size. They always appear to be a lower molecular weight than their actual size, because of their compact structure. Nicked circles generally also appear on the gel when you run uncut plasmids. They run slower and appear to be larger than their actual molecular size. Sometimes linear molecules also appear on the gel, but they tend to be minor species. As indicated above, you need to cut the DNA with restriction enzymes, before attempting to determine the size. If you know the expected sequence, choose a single cutter. If you don't try a few enzymes and add up the sizes of the linear fragments.
As said above, you have to Digest your vector. A usual way is to have three digestions: one negative without enzymes, one with only one Enzyme to linearize your vector and the third one with both enzymes on the both Ends of your insert.
If your insert still gives more intence bands it could rely on Disposition of ethidiumbromid. During my own experiments the bands above 4kbp tended always to be not so intense.
I would do four samples; cut one with enzyme 1, another with enzyme 2, another with both enzymes and a final one with no enzymes. This way you can tell if the enzymes are working properly r cutting more than once in your vector or insert. Run these controls and let us know ablut the patterns you get.
I agree with the statement of Michael R Volkert. The pattern will also be affected by the concentration of agarose. I also suggest you make a single enzyme digestion to determine the plasmid size. Besides, make a double enzyme digestion to get both insert and vector.
You can use a new plasmid extraction kit as well as freshly prepared reagents to verify if your mixture was polluted by any restriction enzyme.
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