I tried extracting DNA from Garcinia fruit and got the following readings with Nanodrop.
Nucleic acid: 149.7 ng/µL
A260/A280: 1.39
A260/A230: 0.12
A260: 2.99
A280: 2.15
Factor: 50
Baseline correction: 340nm 1.27 absorbance
But upon agarose electrophoresis no bands were observed. What could be possible reason for the discrepancies between nanodrop readings and agarose electrophoresis interpretation inferred over here?
Thankjs
Yugendran
the low 260/280 ( should be about 1.8) and the very low 260/230 suggest a lot of protein, very little DNA and even less RNA. You may have reagent carry over leading to inaccurate nanodrop readings as the blank for the nano must be identical to the solvent that the dna is being measured in.. You could try proteinasing the dna sample and re purifying or another dna preparation method but I think that the gel is accurate and you have very little dna and probably a fluorescent band of protein still caught in the gel wells
the low 260/280 ( should be about 1.8) and the very low 260/230 suggest a lot of protein, very little DNA and even less RNA. You may have reagent carry over leading to inaccurate nanodrop readings as the blank for the nano must be identical to the solvent that the dna is being measured in.. You could try proteinasing the dna sample and re purifying or another dna preparation method but I think that the gel is accurate and you have very little dna and probably a fluorescent band of protein still caught in the gel wells
Dear Yugendran
as Paul told you.. dna peak is centered at 260nm and a good dna preparation has to show a 260/280 ratio >1.8.
you need to optimize your dna extraction protocool because are you quantiding something else as confirmed from the absence of dna in agarose gel.
good luck
Manuele
I agree with Paul Rutland suggestion. The lower 260/280 value indicates the poor quality of the DNA. In addition, the DNA concentration also seems lower. These two factors could be the potential cause of "No band" in AGE.
Agree with Paul Rutland. I think, as a procedure, it is better to check agarose gel first before going for nanodrop since you might get some reading for the reagents or proteins that might mislead you into thinking that your sample has DNA.
Agree with previous comments. In Nanodrop for DNA or RNA 260/280 ratio must be ~1.8 or 2.0.Your values are much lower than expected values which indicate a low amount of DNA in your sample which is not detectable. Thus, you are unable to get any band in your gel.
Paul Rutland
hello sir,
You mentioned about a small florescent band of protein in an agarose gel. Can you please explain the chemisty about how EtBr gets intercalated with a protein molecule to yield florescence.
Sarath Nair
https://link.springer.com/article/10.1007%2FBF00782890
Molec. BioL Rep. Vol. 5, 4: 209-214, 1979
A RAPID AND SENSITIVE METHOD FOR DETECTION OF PROTEINS IN POLYACRYLAMIDE SDS GELS: STAINING WITH ETHIDIUM BROMIDE A. VINCENT & K. SCHERRER
I think this area needs a review conventionally; there have always been incongruent in the two estimates, the problem is becoming persistent often.
Hello
Yes your values are much lower than expected ( ~1.8 or 2.0).
The links here will help you
https://www.ogt.com/resources/literature/483_understanding_and_measuring_variations_in_dna_sample_quality
https://www.researchgate.net/post/What_is_the_possible_explanation_for_the_different_outcome_between_Electrophoresis_and_Nanodrop_2000
Good Luck
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