…RNA treated DNA preps and the Nanodrop

This is from the Nanodrop people..”After RNase treatment I would purify the DNA by passing through a column. The free nucleotides themselves will pass through the column (e.g by Qiagen or Sigma), leaving only intact DNA bound, which can then be eluted. I cannot find a definitive answer in the literature, but I would expect the degraded RNA to still absorb at 260nm, therefore I would recommend the additional purification step.”

I don’t know fully if digested RNA fragments are co-precipitated with DNA and hence a column/gel cutting is the only way of separating these 260nm absorbing fragments from the DNA. Anyone got any evidence? 

 Also- in general be careful with preps from columns- there is definitely something ‘weird’ with absorbance profiles samples from the silica plate method (and possibly from other columns). If in doubt use the ‘safer’ quant. method (presuming that Hoeschst dye binds only to DNA).

Anyway…I hope you get the picture!!

One response to “…RNA treated DNA preps and the Nanodrop

  1. Another “safe” method is 0.8 % agarose gel against standard DNA (e.g. lambda) of known concentration e.g. 5, 10, 20, 50 ng ul-1

    This won’t be as precise as nanodrop or the fluorimeter in the robot room *when these are working well* [and isn’t appropriate for very low DNA concentrations and/or volumes], but it will give you:
    (a) an assessment of gross variability in DNA concentration amongst samples
    (b) a rough concentration for each sample
    (c) a visual check on RNA contamination and its fragment length range in BP.
    (d) a check on other contaminants that can bind DNA to agarose, and may interfere with fluorimetry readings

    Compared to other methods, a gel is cheap, robust, and relatively insensitive to the method of DNA extraction

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