Fuzzy bands on agarose gel after ligation - (Oct/02/2006 )
Hi there,
After I perform ligation reactions, I always run a sample on a 1% agarose gel to make sure it worked. However, I frequently get a diffuse band around the appropriate size, rather than a crisp band.
Does anyone know what causes this, or if I could still use that product as a template for PCR?
Thanks!
why are you using a ligation reaction mix as a PCR template?
Because I have three fragments to assemble that I'd like to amplify up by PCR before inserting into my vector for transformation.
you don't worry about confirming the identity of your clone before moving on?
Well yes, I will. Right now I am just attempting to assemble the vector, which I will transform into XL-1-Blue, isolate the plasmid again, then verify it is has the correct insert via PCR using primers to the two outermost fragments.
My big question is just for ligations in general, why one would get a fuzzy/diffuse band as opposed to a crisp one.
that is the million dollar question and speaks to the difficulty of cloning, and why cloning is a form of art... 
I would also confirm the clone by direct sequencing using vector-derived sequencing primers. it is rarely cut-and-dried and you often can get multiple products. please don't take my word for it; browse this site and you'll find many many threads where PCR-confirmed clones did NOT yield the proper insert upon sequencing. there are many possible reasons: recombination, degradation of ends, lack of specificity in the initial PCR, mistakes in primer-design, ligation of fragments and concatamers...the list goes on. it would surprise me more if you got a good crisp band every time 
I suppose there is the possibility in this instance that your blurry bands are purely mechanical and have to do with the gel (ran too fast, incorrect buffering, problem with sample, etc) but it is more likely that your ligation mix contains more than one specific product in that little tube
I would also confirm the clone by direct sequencing using vector-derived sequencing primers. it is rarely cut-and-dried and you often can get multiple products. please don't take my word for it; browse this site and you'll find many many threads where PCR-confirmed clones did NOT yield the proper insert upon sequencing. there are many possible reasons: recombination, degradation of ends, lack of specificity in the initial PCR, mistakes in primer-design, ligation of fragments and concatamers...the list goes on. it would surprise me more if you got a good crisp band every time
I suppose there is the possibility in this instance that your blurry bands are purely mechanical and have to do with the gel (ran too fast, incorrect buffering, problem with sample, etc) but it is more likely that your ligation mix contains more than one specific product in that little tubeNothing more to add, Amikins has the story spot on. Blurred bands boils down to mechanical, (overloaded gel, too much salt, or gel run too fast) or biochemical (a mix of DNA fragment of similar size) or a combination which sometimes happens with PCR.
To get decent bands at all from a ligation mixture you must heat kill the ligase. Don't do this with PEG containing ligation buffers (most of the "quick ligation" kits), since this trashes the DNA in a way I don't understand -- but it doesn't work.