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Does DNA have to be supercoiled for transient protein expression?


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4 replies to this topic

#1 scichem

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Posted 02 February 2012 - 12:39 PM

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!

#2 bob1

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Posted 02 February 2012 - 12:48 PM

Either will work, however supercoiled DNA transfects more efficiently than circular or linear DNA.

#3 scichem

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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 bob1

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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 scolix

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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.




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