I am observing a band at 20kb after ligating a 1.5kb insert with a 4.4kb pCold I plasmid, which should ideally result in a band around 5.9kb (assuming successful ligation). I used BamHI-HF (NEB) and Fast Digest Hind III (Thermo Scientific) enzymes for digestion, and observed similar results with the combination of their buffers. Could this be causing the issue? How can I ensure a band of approximately 6kb is observed?
Are you referring to the size of the DNA after ligation, or the size of the DNA from mini preps after transformation? If this is your ligation product ,then just don't worry and go ahead and transform it.
If this is what you see from your mini prepped DNA after transformation then there is clearly some issue. A picture would help because there are many possibilities but I doubt it has anything to do with buffers.
Michael J. Benedik Yes, I am referring to the size of the DNA after ligation. Thank you for the reassurance.
You get all sorts of forms with ligation but the cell usually sorts them out after transformation.
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