I am doing an Nde1 and BamH1 double digest. The gel is as followed Pet-28a with no insert (undigested), pet-28a (digested),Thermoscientific zip ruler express 2, pet-11a with insert (undigested), pet-11a with insert digested.
Is the presence of two bands in the undigested lanes because of supercoiled or linear, nicked etc plasmid? Why do I have at least 4 bands in the digested lanes? I presume the digestion was incomplete but am uncertain as to how to explain all the differences and bands I am seeing? The very faint bad at the bottom of the fifth lane at approx 700 bp does correspond to the size of my insert of interest.
Gary Robertson
Do you know how many bands you are expected to see? And why do you assume its incomplete digestion? there is a clear difference in the banding pattern for your plasmid without insert and for your plasmid with insert and so would appear to me based on the info you have given that your digest was successful, yielding the insert @~700bp.
If it is that you are expecting more bands in the pet-11a digest, ensure that the expected bands are not too small because it might be that they ran off the gel
The plasmids to my knowledge have only one Nde1 and bamh1 recognition site. I therefore expected to see the plasmid and the insert.
Seeing that the banding pattern of pet28a and pet11a digested is similar except for the insert seen for pet11a, then it could be that the plasmid has more Nde1 or Bamh1 sites than is thought to be the case.
Be carrefull with the star activity of BamHI. Try a rapid digestion with less unities of enzymes
Test for incomplete digestion and the potential presence of (5')- or (3')- hydroxylated versions of the oligos (RNA or DNA) versus the (5')- or (3')- phosphorylated counterparts. For a relative interpretation of a similar nucleic acid behavior you may see ref (http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-61779-270-0_2) where we observed triplets of protein-bound nucleic acids (e.g., ADP-Ribose oligonucleotide chains) for the first time back in the 80"s as first reported on my doctoral dissertation since there was a combination of phosphodiesterase + 5'-phosphatase presence on a permeabilized cell system.
I encountered with the same situation .And I only do an Nde1 and BamH1 double digest for a pET28b(+)-SUMO vector. And I was expecting only one band around 5600bp. However, I got multiple bands after gel running.
I did over night digestion at 37 degree. I checked the information about the construct, there was only one Nde1 and bamh1 recognition site. Can anyone help me with this?
I think you may have experienced star activity, where the Enzyme cuts sequences which are not the ones it should recognize. Where did your enzymes come from and what star activity do they report? Otherwise I would suggest a shorter incubation. Overnight at 37 degrees is much more than is usually necessary.
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