I am trying to clone a 7.3 kb insert into a plant binary vector which is around 7.2 kb. The enzyme that I am using is PacI which has 2 bp overhang. I used electroporation and the cell that I used for electroporation was Bioline ElectroShox competent cell. I screened 20 colonies by colony PR and found 5 positive. Then I miniprep two of them using Wizard plasmid purification kit (Promega) and digested with couple of enzymes. With every digestion I am getting the right size band as well as several unexpected band (photo attached). Can anyone explain what is going on?
Why are you restricting with a couple of enzymes? Should you not be confirming the cloning by restricting the plasmids with the same RE that was used to prepare the vector and insert? Hopefully you are using the REs with appropriate buffers.
The sum of the stoechiometrically appearing bands seems to be around 14.5 kb, but there are additional uncut bands at 2.5 and 1.4 kb with differing copy numbers, likely not belonging to your plasmid.
How is the undigested plasmid look on gel? Are these enzymes sites, which you used, inside the insert or vector? I suggest you to take a site which is only in vector or only in insert and do a single digestion, that will tell you the exact size of the plasmid. I am not sure but its a little difficult to resolve 7.2 from 7.3 kb on your present gel.
Dear Hassan,
The vector and insert is coming around same size and reaching 14kb after ligation. Have you checked the site (NcoI, XbaI, BamHI) in vector as well as insert? Since you have cloned at PacI site, insert can be either orientation with respect to promoter. Still I feel, your digestion is not proper when compere to uncut.
These all bands are present in uncut lane so I think they are not due to any error in cloning but it could be due to poor quality of plasmid that you purified from kit.
in my suggestion use different kit or repeat the experiment as the quality of DNA improves these band will disappear
your uncut sample is very suspicious and interesting!!!! you have bands of 1.4 and 2.5kb in all samples as in your uncut! 1.4 is sharp in all of them and 2.5 faint! how can it be existing without digestion ofcourse if your plasmid is ok ..
strongly I believe you have contamination in your extracted plasmid... having those unwanted bands in your UC means that they are already existing in your plasmid! it has been contaminated by your extraction kit or water or some where else... ONLY after digestion by XhoI you have your desired band of 14 kb linearized vector (I hope there is only one site of XhoI and that is the linear band of 14kb) so change your extraction buffer or conduct another extraction and bacteria using the same kit to check your extraction kit components...
also check the digestion manuscript of the enzymes to avoid partial digestion or star activity of the enzymes for the digestion of the 1-4 lanes...
so as conclusion from my point of view you have bands of 1.4 and 2.5 because of the contamination and other bands because of the unsuccessful digestion...
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