Home FAQs Why are the DNA ladders showing up on my Southern blot? What is the sequence or composition of the ladder bands?

FAQ: Why are the DNA ladders showing up on my Southern blot? What is the sequence or composition of the ladder bands?

This phenomenon has been observed in a variety of labs, and, while we do not have an absolute explanation, there are 2 likely sources for the seemingly unlikely hybridization of the user’s probe to the ladder’s DNA. One is that, if the probe itself was generated from a pUC or pBR based plasmid, there may be a low level of plasmid contamination in the probe that is hybridizing to the ladder’s bands, most of which have pUC-like sequences in them.

Alternately, there may just be some random, low specificity homology between the probe and some segment of the ladder fragments. The bands consist entirely of DNA derived from either pUC or Adenovirus 2 DNA, which is used as filler DNA to generate plasmids of the right size for the markers. Because the molar amount of DNA in the ladder bands is frequently in great excess compared to the DNA being probed, even a relatively low homology can still result in enough incorporation of probe to give a readily detectable signal.