I am doing protein – Linear pUC18 DNA complex studies and further trying to resolve the complex on the Superose 6 column. The following information is provided for your clarification
- On AKTA pure both A280 and A260nm wavelength are activated during run, but I could not find a minimum of 0.1mAU even through out the column volume (24mL)
- The concentration of pUC18 DNA in reaction complex as well as LpUC18 alone is from 0.1 to 1.5ug
- The salt concentrations in the reaction are from 150 to 500mM
- Linear pUC18 DNA mwt is ~1740kDa so there is no doubt about resolution problem or elution at void volume on Superose 6 as its fractionation range falls in the range of 5kDa to 5mDa.
But the question is I am not able to detect the Linear pUC18 DNA either alone or in the complex. What could be the possible reason not to detect the pUC DNA? Or is it may be the concentration of pUC18 DNA is not sufficient to detect on A260nm? Is anyone has experience with linear pUC18 DNA separation on Superose column? Your valuable suggestions and comments are most appreciated.
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