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I don't understand why three bands are found without any restriction digest. Please answer me as soon as possible.

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  • $\begingroup$ today, i have seen fourth band first time after plasmid(pNL4-3) isolation $\endgroup$ – Kumaraswami Chintala Oct 10 '16 at 14:02
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These are the three forms of the plasmid: supercoiled (smallest 3D-structure, runs fastest), linear (runs in the middle) and relaxed, nicked plasmid (runs highest). You can have another fourth band, which is the circular single stranded plasmid (runs faster than the supercoiled version). See the following figure (from this article, which I recommend reading):

enter image description here

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  • $\begingroup$ Thank you a lot. I have another question, How many DNA fragments are generated from a circular plasmid DNA, when this DNA is restricted by EcoRI three times and each of these EcoRI fragments is restricted one time by PstI? $\endgroup$ – Rashid Jun 14 '14 at 20:38
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    $\begingroup$ Can you please make this a new question? And if an answer is satisfactory answering your question, can you please accept it? This is a nice gesture towards the people who anwer. $\endgroup$ – Chris Jun 15 '14 at 0:12
  • $\begingroup$ Thank you Dear Chris. I accepted your ansewr. I got confused again. I found in wikipedia that relaxed plasmid(mentioned that both strand are uncut) is different than open circular(one strand is open). But here both are considered as same. In case of second question, Already I asked as a different question and I didn't get any answer. waiting for response. Thank you $\endgroup$ – Rashid Jun 15 '14 at 15:01

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