Question about running plasmids and restriction enzymes

[Really Lost]

Hi

 

I ran a gel with plasmids digested by a restriction enzyme. The cut plasmid is in three seperate bands at 4000 bp, 3000 and 2000

 

I also ran a control with the plasmid without a the restriction enzyme in another well of the gel. It has moved further past the first cut band to about 3500 bp? Is this possible because the plasmid is coiled and the cut pieces of the digested plasmids are more linear? Or is this not possible.

 

There seems to be two bands in my control which has me worried. the other which much thicker could be RNA I suppose. right?

 

 

Thanks

[CharonY]

Uncut plasmid DNA runs as supercoiled and will therefore run faster. You would have to linearize your plasmid first for correct size estimation. In most plasmid extractions you would include an RNAse step. But even without RNA rarely resolves as a clear band but rather than a smear towards the bottom.

 

Plasmid DNA is usually seen in several conformations including open circle (single nick) as well as multimeric forms. Sometimes chromosomal DNA also gets co-purified.

Edited March 23, 2012 by CharonY

[Really Lost]

Thanks for your response.

 

One other thing, I ran a plasmid called pUC18:URA3 with HindIII and got 4 fragments. However I don't know if that is correct. I looked up pUC18 and it doesnt seem to have any restriction sites except for at the Multi Clonal Site. So I am wondering how is it possible to have that many fragments. I think the URA3 fragament that is attached to the plasmid only has one hindIII site. not sure though. I thought there might be a problem with the enzyme or the buffer.

 

How can I find out for sure how many HindIII restriction sites it has (and therefore how many fragments I should get)?

[CharonY]

You can make a rough assessment based on the known sizes. pUC18 alone should yield a linear fragment. How big is the URA3 insert and if all the other sites in it, is it theoretically possible to yield the sizes you see? The accurate way is to get hold of the whole sequence and just check for restriction sites. But first make sure that the restriction actually worked.