I run 0.7% agarose gel electrophoresis of my plasmid DNA sample consisting of GFP vector using NheI and HindIII restriction enzyme. I used two types of plasmids, i.e., isolated by mini prep-alkali method, and another isolated by midi prep system (pure) of the same source E. coli. the 4 & 5 lines show cut and uncut plasmid respectively isolated my midi prep system. The 1, 2 & 3 show the 500 bp marker, cut and uncut of mini prep isolated plasmid signal respectively. In the attached image, we found extra band in the plasmids isolated by alkali method (line 2 & 3), but in the pure samples (line 4 & 5) no such extra bands found. What are the possible reasons behind the result? How can I interpret this result? If any one experienced, please share.
Hi Mahmoudpour,
Thank you for sharing valuable opinion. Here is my experiment condition. Please comment on it.
The concentration of the pure solution is 126 ng/ul, and the other plasmid conc. is unknown. I used 2.5 ul water,1.5 ul of plasmid with 0.5 ul buffer, and 0.25 ul of each enzyme. The buffer composition is okay with both enzymes. Then reaction took place for 30 min at 37 'C temp and electrophoresis run for 25 min.
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