I have included pictures to illustrate my point. In the gel image that has only 4 lanes: Lane 1 is a 1 kb molecular weight ladder. Lane 3 is my wild type DNA (in a pGEX plasmid) and Lane 4 is the same DNA, which has undergone mutagenesis to include a single nucleotide substitution. This is not a great resolution plasmid extraction, but I have included it to demonstrate that the wild type looks different than the mutated DNA. This cannot be an anomaly, because I have seen this same result on numerous occasions (refer to the second gel images, which shows the plasmid extractions from five different wild type samples). I am completely at a loss regarding these results. Any help will be greatly appreciated.
Do you think that because you have changed the sequence that the smear is a result of the structure flipping between coiled and supercoiled conformations because neither is completely stable at the temperature generated in running the gel so you are seeing both structures and some transient intermediate conformations ? If this is the case then running the gel at lower voltage in a cold room or using cooled buffer may give a better defined image
Thank you very much for all the input. I have realized what the problem was; I transformed the plasmids into BL21 condon plus cells, which have a plasmid of their own conferring chloramphenicol resistance to them. So the additional 3 bands that I was seeing was the BL21 plasmid bands. I confirmed this by performing another gel electrophoresis today and I saw the same results for my mutated plasmids. I will propagate my plasmids in DH5a cells instead going ahead.
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