We have made total DNA extraction and seen absorbance value in nanodrop, but, we couldn't have a image from gel electrophoresis, and consequently, we thought that DNA was degenerated. Could you give me some information about this problem?
Hello Pelin,
This could have several reasons.
First, a contamination of the nanodrop that yielded a false positve reading with your sample?
Second, a total DNA extraction of what? If it's genomic DNA for instance, and if you haven't digested it, it's normal to see nothing on agarose gel because all genomic DNA remained in the well, and you see nothing on the lane.
Other reasons ?
Best regards,
Philippe Urban
Hello Pelin,
This could have several reasons.
First, a contamination of the nanodrop that yielded a false positve reading with your sample?
Second, a total DNA extraction of what? If it's genomic DNA for instance, and if you haven't digested it, it's normal to see nothing on agarose gel because all genomic DNA remained in the well, and you see nothing on the lane.
Other reasons ?
Best regards,
Philippe Urban
Search for the nanodrop on-line technical notes - they describe a lot of things that can affect nanodrop readings including some of the reagents you can use to extract DNA (see the following extract) and are a good way of really understanding what the readings tell you and how to troubleshoot.
"Residual chemical contamination from nucleic acids extraction
procedures may result an overestimation of the nucleic acid
concentration and/or negatively influence downstream
analysis. Shown below (fig. 1) are example spectra for 4
common extraction reagents which, if not properly cleaned up,
will affect sample purity.
FIGURE 1. Spectra of reagents used in the isolation of nucleic acids.
A) TriZol B) Phenol C) Guanidine HCL and D) Guanidinium
isocyanate"
Dear Urban,
I don't have any information about be calibrated of nanodrop (in our lab's), or not.
My genomic DNA samples from plant material. And, I need to some information concerning all the time genomic DNA samples never seen band in gel electrophoresis results?
Thanks your answer.
Pelin Turhan,
After DNA extraction if you got the absorbance in nano drop, then this absorbance is because of following reasons.
1. presence of nucleic acid, both DNA as well as RNA
2. presence of phenolic substances
If nucleic acid is there, then you should get DNA and/or RNA band in agarose gel.
If you don't see anything in gel and due over harsh treatment, nucleic acid got denatured then the absorbance is due to the hyperchromic effect.
Dear Rees,
I compared results of nanodrop measurement and i saw difference between absorbance value of my extracted DNA samples, for instance, if one experiment's absorbance is 1.90 to 1.99, other ones 2.3 to 2.7 which wasn't make treatment with RNAse A. And also my problem is about can't understand nanodrop a reliable source of information for DNA quality/concentration?
Thank you for your interest.
As pointed earlier, make a gel with digested genomic DNA and see what comes on your Etbr staining.
You can also do a test experiment with a "known" plasmid DNA like pUC18 or any other. Measure in the nanodrop, digest with one or more enzymes (in parallel a control without any enzyme) and run them on the gel to see whether the DNA is comparable with your nanodrop read out. If it work, you can also try doing with 2-3 difference concentrations to prove it.
good luck!
The last step in DNA extraction, purify your DNA very carefully some time some chemicals remain present in DNA after purification.
secound, set voltage very close according to length of electrophoresis chamber.
good luck!
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