distinguishing circular and linear DNA

[cmgross]

I need to learn at least 2 different ways to determine if a 100 kb ds DNA viral genome is circular or linear. We are starting with highly purified viral DNA in a tube. I know that circular and linear DNA run differently on a gel, but I don't think you can run fragments that big on an agarose gel. Thanks

[canalon]

Pulse field gel electrophoresis (PFGE) is your friend

[JackBean]

By restriction enzymes you could recognise it (circular = you will get as many fragments, as many times you cut; linear = you get +1 fragment), but I guess ou don't have restriction map, do you?

[qqsvery]

U can try density gradient centrifugation method！good luck！

[michellethemit]

Disregard this, the method I was going to suggest required running on an agarose gel, and the OP mentioned that he was unsure whether a 100kb size genome would run properly.

[Darwin420]

Yea, pulse field is indeed your friend of big samples, if you do this you should be able to differentiate a circular from a linear DNA sample.

Or set up a density gradient centrifugation.

I can't think of any more really.

[gradkid]

Run 2 restriction digests with 2 different restriction enzymes, followed by a combination digest. Let's say enzyme A cuts your DNA into 3 fragments and enzyme B cuts it into 4. You don't know if your DNA is linear with 2 restriction sites for A and 3 sites for B or circular with 4 sites for A and 5 sites for B. However, if you run a combination digest with enzyme A+B, you can determine if it's linear or circular. If the combination digest has 7 fragments, it's circular; if it has 6 fragments, it's linear.

I think.

[aptitude]

Pulse-field gel electrophoresis is good for large DNA fragments.

[greatmicrobiologist]

Disregard this, the method I was going to suggest required running on an agarose gel, and the OP mentioned that he was unsure whether a 100kb size genome would run properly.

Support you.

@Cmgross:

before going for agarose gel elctrophoresis or polyacrylamide gel electrophoresis, go for PCR amlification for your desired quantity.

[JackBean]

Pulse-field gel electrophoresis is good for large DNA fragments.

yeah, that's what the first reply suggested

Disregard this, the method I was going to suggest required running on an agarose gel, and the OP mentioned that he was unsure whether a 100kb size genome would run properly.

Support you.

@Cmgross:

before going for agarose gel elctrophoresis or polyacrylamide gel electrophoresis, go for PCR amlification for your desired quantity.

Why should he run PCR? How will that help?