The mysteries of Nanodrop - (Dec/23/2005 )
Hello everyone 
I´m one of the poor guys who tries to establish chromatin immunprecipitation.
One problem here is, that the DNA concentration after immuneprecipitation is
usually very low. In my last run I measured around 17 ng/µl with immuneprecipitates
and ~ 250 ng/µl of my whole cell extracts in 10 mM TrisHCl pH 8.0 (with no immuneprecipitation).
The Nanodrop manual gives me a lower detection treshhold of 1,5 ng/µl, which I don´t believe.
But can I trust values around 10 - 20 ng/µl? Furthermore the 260/280 values were quite strange:
with the WCEs (250ng/µl) I got something around 1,85 which should be fine for DNA, but for my Immunoprecipitates I got ~ 1,56, which means some protein contamination. But the procedures for DNA extraction and precipitation were exactly the same (including one Phenol/C/IA extraction step with phase lock tubes, the aquatous phase was not milky afterwards). So I can´t imagine any protein contamination. Can I trust these low numbers and can I at least say, I have some
DNA in my samples at all?? ( The spectrometer curves had the amplitude at 260 and were not kind of shaking) I would be most grateful, if someone could help me out
sorry..I cant help you with this..but just because i will have to set up ChIPs soon...can u please tell me in detail which protocol you are following...
thanks a lot..I would really appreciate it
I´m one of the poor guys who tries to establish chromatin immunprecipitation.
One problem here is, that the DNA concentration after immuneprecipitation is
usually very low. In my last run I measured around 17 ng/µl with immuneprecipitates
and ~ 250 ng/µl of my whole cell extracts in 10 mM TrisHCl pH 8.0 (with no immuneprecipitation).
The Nanodrop manual gives me a lower detection treshhold of 1,5 ng/µl, which I don´t believe.
But can I trust values around 10 - 20 ng/µl? Furthermore the 260/280 values were quite strange:
with the WCEs (250ng/µl) I got something around 1,85 which should be fine for DNA, but for my Immunoprecipitates I got ~ 1,56, which means some protein contamination. But the procedures for DNA extraction and precipitation were exactly the same (including one Phenol/C/IA extraction step with phase lock tubes, the aquatous phase was not milky afterwards). So I can´t imagine any protein contamination. Can I trust these low numbers and can I at least say, I have some
DNA in my samples at all?? ( The spectrometer curves had the amplitude at 260 and were not kind of shaking) I would be most grateful, if someone could help me out
I use the Nanodrop. I have been using for low dilutions for the purposes of diluting transgenic cassettes to 5ng/ul prior to submission to the microinjection facility. I have found the actual reading seems to accurate but what I find with gel purified products (using GeneClean Spin Kit)that have a low concentration is that the 260/280 reading tends to be a useless reference as the value makes very little sense. The microinjection facility never came back to me saying the concentration I stated was incorrect and the mircoinjections have always led to at least five positive founders per cassette.