This type of problem has been know for many years. If you do a google search on ghost band plasmid you get these papers which discuss the problem, show pictures of the types of bands and suggest why they arise as well as how to get rid of them/stop them appearing in the first place.

For a genera discusion see: Trends Biochem Sci. 1996 Nov;21(11):441-2.

Methods and reagents. Eliminating ghost bands from plasmid preps.

http://www.ncbi.nlm.nih.gov/pubmed/8987401

and for examples see

Anal Biochem. 1996 Oct 15;241(2):186-9.

Identification and eradication of a denatured DNA isolated during alkaline lysis-based plasmid purification procedures. http://www.ncbi.nlm.nih.gov/pubmed/8921185

Hi;

My first thought would be star activity, but since you only saw one extra band, I'd probably dismiss that thought pretty quickly. My second thought would be incomplete digestion, which can happen if your enzyme isn't working at peak efficiency and/or you weren't digesting long enough. I've found that this results in bands of sizes I didn't expect to get because the DNA is just stuck in various degrees of supercoiling and digestion. In these cases, I add a bit more enzyme and repeat the digest and that usually clears up the extra band. If that doesn't work, then like Pedro, I'd begin to think that you had a heterogeneous population of plasmids, in which case, I'd want to test with either a panel of restriction enzymes or a panel of primers (PCR and check for product sizes).

Hope that was helpful,

Ebony

I would vote for partial or incomplete digestion. Without knowing more specifics about your vector and number of cuts expected. You could run a little undigested plasmid next to your restriction digestion, and if you see the same size ghost band, that would suggest that it is uncut plasmid (perhaps supercoiled).

hi sarah....we also face such things. sometimes the vector is too supercoiled to open up and get digested...and they run around 2-3kb (original size being 5-6kb).

To confirm ur clone, try to put different set of digestions (internal/external) with other enzymes. Keep ur restriction digestions for 3-4 hrs at 37C) and heat inactivate ur reactions at 65C for 15mins.

If everything looks fine..just go ahead with further experiments...dont bother for ghosts...

Do reply if u sort it out.

all the best

I'd concur with the above suggestions, but would ask, does the original uncut supercoiled plasmid DNA run at, or just a little higher, than the 'ghost band' that you're seeing? You may have denatured-supercoiled DNA, which will tranform, but will not cut with restriction enzymes. I'd also suggest that the DNA Andaleeb described as 'too supercoiled to open up' is also denatured-supercoiled DNA.

However, if this is commercially bought vector (and thus, presumably, >95% supercoiled), rather than from your own plasmid prep (which may have nicked OC, SC and SCd species), I'd but my bets on partial digest.

Hi All,

I have uploaded my results so far. The first is of the gels is my "bought in" vector and Jamie Caryl you could be right that it is caused by my plasmid prep, I bought it in the format of ready transformed bacteria. I hope because of this that it is unlikely I have contaminants. I am using this for mammalian cell transfection so I would need it as "clean" as possible. Scott Diede I have included the undigested plasmid on one of my gels, the band is a little lower than that and interestingly appears in the non digest too. So as you have all suggested is probably the vector in a different state of supercoiling, possibly caused by the plasmid prep.

I extracted the empty vector from the first digest and then religated to make my control. The second gel is testing that my empty vector is present and correct. Again I have ghost bands there but not as prominent. I had trouble finding compatible enzymes that were not really expensive. I opted for SMAI and HINDIII because those enzymes were compatible and some we had in. However the double digest for the empty vector only has one band as the resulting product from these 2 enzymes leads to a very small fragment. I am going to test another set that will lead to 2 clear bands.

Thank you all for your advice :)

That is denatured DNA, and you can see from the other plasmid prep (of the vector with gene) that there is a wee bit here too, but crucially it is proportional to the size of the plasmid (it runs a little higher than the denatured of the empty vector). If you'd seen the exact same band across all your preps this may have been evidence of a single contaminant.

If you are working with a standard cloning strain of E. coli and doing mini-preps, I would literally neutralise the alkaline lysis step after two or three inversions (

This type of problem has been know for many years. If you do a google search on ghost band plasmid you get these papers which discuss the problem, show pictures of the types of bands and suggest why they arise as well as how to get rid of them/stop them appearing in the first place.

For a genera discusion see: Trends Biochem Sci. 1996 Nov;21(11):441-2.

Methods and reagents. Eliminating ghost bands from plasmid preps.

http://www.ncbi.nlm.nih.gov/pubmed/8987401

and for examples see

Anal Biochem. 1996 Oct 15;241(2):186-9.

Identification and eradication of a denatured DNA isolated during alkaline lysis-based plasmid purification procedures. http://www.ncbi.nlm.nih.gov/pubmed/8921185