Electrophoresis Problem-cut versus uncut

[agentnerdo]

Hello,

 

 

I ran a electrophoresis for a intro bio university class. Each group was given two ladder, and two wells, one to use with ECOR1+DNA and one with just the DNA.

 

 

 

One group came up with a question that stumbled me;

 

they forgot to put their ECOR1 in their DNA (or just didnt give it enough time to do its magic), but now they have two strands with the same length.

 

The reason I feel the need to help them is because the ladder I filled leaked into their wells, and they ended having three lines show up, two perfectly matching the ladder, and one perfectly matching the uncut. This resulted in them having a rather hard time figuring out how they could get three lines with the furthest one being as far as the uncut.

 

 

Now for the question, is their anyway to figure out what the unknown is using the uncut?

 

I personally already calcualted the equation to count for bp, but I know that this does not apply to uncoiled, since the equation is based on the ladder, which is linear.

 

The unkown can either be pUC 19, pDrive, or pGen easy,

 

 

any comment be appreciated!

[CharonY]

Just to validate the setup: you got four wells. Two with ladders, one with cut and one with uncut DNA, correct?

 

Your goal was to differentiate between three different plasmids by using restriction enzymes, correct?

 

So in one of the wells there are three bands, two corresponding to the standard and one corresponding to the uncut DNA?

In other words you do not have restriction (the fact that marker got into the well is not of interest here) and essentially two well showing the same uncut plasmid?

 

Now as you assumed correctly supercoiled DNA runs faster than linear DNA so you cannot easily deduce the original size of the plasmid. Well in theory you could, but you would have to run the uncut plasmids in a gel (with a given percentage, probably around 0.8-1%) and then compare it with the marker. But if you do not have all the plasmids on the gel, i.e. only the unknown, you are lacking a point of reference here.

pUC 19 is around 2.6 kb, pDrive 3.8 and (I assume) pGEM-T easy is around 3kb. So even then it will be tricky to differentiate between pUC19 and pGEM, unless you run them together.

So in other words, unless the gel looks terrific and you know precisely the run length of the supercoiled plasmids (which I assume you do not, considering the question in the first place) I believe you need at least a gel with all the plasmids or even better, retry the restriction.

 

If, on the other hand the uncut plasmid is different from that one you want to identify, it may be possible to assign a tentative identity (ideally the reference plasmid would have been pGEM).

[agentnerdo]

thnx allot for your input,

 

in summary i doubt its possible distinguish between the three unkown, since only one unkown was used in the gel

 

had we used all three...we should be able to distingish between them