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distinguishing circular and linear DNA

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distinguishing circular and linear DNA

Postby cmgross » Sat Oct 31, 2009 3:49 pm

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
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Postby canalon » Sat Oct 31, 2009 11:29 pm

Pulse field gel electrophoresis (PFGE) is your friend
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Postby JackBean » Sun Nov 01, 2009 12:13 am

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? :)
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Postby qqsvery » Sun Nov 01, 2009 2:31 pm

U can try density gradient centrifugation method!good luck!
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Re: distinguishing circular and linear DNA

Postby michellethemit » Sat Dec 12, 2009 8:06 pm

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.
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Postby Darwin420 » Mon Dec 14, 2009 5:42 am

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.
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Re: distinguishing circular and linear DNA

Postby gradkid » Thu Aug 25, 2011 2:00 am

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.
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Postby aptitude » Sun Sep 11, 2011 8:41 pm

Pulse-field gel electrophoresis is good for large DNA fragments.
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Re: distinguishing circular and linear DNA

Postby greatmicrobiologist » Mon Sep 12, 2011 12:22 pm

michellethemit wrote: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.
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Re:

Postby JackBean » Thu Oct 20, 2011 8:18 am

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

yeah, that's what the first reply suggested

greatmicrobiologist wrote:
michellethemit wrote: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?
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