I purified my DNA using a Qiagen Maxiprep kit, and I have two bands on my agarose/EtBr gel. I'm assuming that one is supercoiled, while the other may be linear. For transfection and expression of protein, is the only usable DNA the supercoiled portion?
Thanks!
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Does DNA have to be supercoiled for transient protein expression?
Started by scichem, Feb 02 2012 12:39 PM
4 replies to this topic
#2
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Hmmm, I think it's working
Posted 02 February 2012 - 12:48 PM
Either will work, however supercoiled DNA transfects more efficiently than circular or linear DNA.
#3
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Posted 02 February 2012 - 12:54 PM
What could be happening that would cause my dna to be circular/linear, instead of supercoiled? How do I maximize the supercoiled form?
#4
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Hmmm, I think it's working
Posted 03 February 2012 - 09:23 PM
It is pretty much impossible to get a prep that is 100% supercoiled; there is almost always some degradation during extraction. Much of this degradation comes from shear forces in centrifugation steps and from chemical digestion during the alkaline step.
#5
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a student
Posted 04 February 2012 - 11:02 AM
I agree with bob1. You will always have supercoiled DNA along with some nicked DNA.
DNA from maxi prep should give you a good percentage of supercoiled DNA. And be gentle.
DNA from maxi prep should give you a good percentage of supercoiled DNA. And be gentle.
