Its the first time I work with plasmid pEGFP-N1 but when I runned the gel agarose get two band and my band mini preparation is low. Please give me suggestion why I get two band? thanks very much.
Here my result running gel agarose
If you ran a plasmid sample without restriction digestion (1-cut), it is very likely to get >1 bands on an agarose gel. This is due to plasmid supercoil (give smaller size on agarose gel) or dimerization.
Is there possibility plasmid DNA due to long time stored and the conformation plasmid became single strain?
because I used uncut plasmid, and long time stored so the plasmid have changed the conformation became nicked, linear and supercoiled. Could it like that?
It has to do with the intracellular enzymes of bacteria. This is addressed on page 3-4 in this link : http://delliss.people.cofc.edu/virtuallabbook/LabReadings/Miniprep/MINIPREPARATION.pdf
Dear, Mr Huang King Kong
Now, I have done twice large preparation plasmid pEGFP N-1 uncut, using alkaly lysis, and continue purification with CsCL density gradient centrifugation by using rotor VTi 90, 65.000 rpm, 16 hour 20 Celcius, but I didnot get band DNA. Could you mind help me, why did not get band DNA? Thanks very much
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