- October 31, 2009 at 3:49 pm #12151cmgrossParticipant
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
- October 31, 2009 at 11:29 pm #94288canalonParticipant
Pulse field gel electrophoresis (PFGE) is your friend
- November 1, 2009 at 12:13 am #94290JackBeanParticipant
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? 🙂
- November 1, 2009 at 2:31 pm #94298qqsveryParticipant
U can try density gradient centrifugation method！good luck！
- December 12, 2009 at 8:06 pm #95894michellethemitParticipant
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.
- December 14, 2009 at 5:42 am #95974Darwin420Participant
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.
- August 25, 2011 at 2:00 am #106045gradkidParticipant
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.
- September 11, 2011 at 8:41 pm #106295aptitudeParticipant
Pulse-field gel electrophoresis is good for large DNA fragments.
- September 12, 2011 at 12:22 pm #106307
- October 20, 2011 at 8:18 am #107051JackBeanParticipantquote aptitude:
yeah, that’s what the first reply suggestedquote greatmicrobiologist:
Why should he run PCR? How will that help?
- You must be logged in to reply to this topic.