I am purifying a PCR product using Quiaquick gel purification protocol. I began with a high concentration of amplified DNA segment- I verified my results using both gel electrophoresis ( to verify the length - ~400 nt) and also ran a nanodrop - which indciated that I had pure DNA. I then ran the Quiaquick Gel purification protocol - I did use Isopropanol, but only washed - not wanting to loose any of my DNA. After I finished the protocol I ran a sample of the purified product on a Gel. The bands from the Gel of the purified product matched the results from before- indicating that I shoudl have successfully purified my DNA product. However, I also ran a sample of my purified porduct thorugh the nanodrop spectrophotometer, which indicated that I had pure RNA- not the DNA I thought I had purified/ started with. Does anyone have an explanation for why this may have happened? Could this be caused by the isopropanol or perhaps by not washing the DNA enough to remove something that could skew my results? Any tips on how to ensure this does not happen again?
Please, never trust the spectrophotometer, when you see nice gel! Any kind of contaminants can make your spectrophotometer measuring crazy. I saw the values minus 0.1-0.5, having nice DNA bands (verified by 3 spectrophotometers). Both DNA/RNA have max. absorbance at 260 nm, thus it is very possible the mistake is there...
Best - Jan
Please, never trust the spectrophotometer, when you see nice gel! Any kind of contaminants can make your spectrophotometer measuring crazy. I saw the values minus 0.1-0.5, having nice DNA bands (verified by 3 spectrophotometers). Both DNA/RNA have max. absorbance at 260 nm, thus it is very possible the mistake is there...
Best - Jan
I agree with Jan. If you still suspect this, add a pinch DNAse and/or RNAase, incubate a bit and run the gel. Sample carrying DNase must for smear.
Nanodrop is not always concrete especially if it is not calibrated it. Trust your gel as Jan said
I definitely agree with Jan. You cannot completely trust nanodrop and if you see a nice well defined band in your gel you shouldn't worry. Maybe you have some residual leftovers of the buffers you used for the purification that make the absorbance ratio indicate RNA or the nanodrop is not properly calibrated. It's not unusual. I would follow withe the purified PCR product keeping in mind that the concentration given by the nanodrop can be somehow inaccurate.
I agree with above trust your gel, we tend to qualify all our nanodrop readings using q-bit for DNA and agilent bioanalyser trace for RNA. They are often quite discrepant. See if you can get your nanodrop calibrated or try a different nanodrop spectrophotometer.
Gel purification relies on dissolving agarose to smaller oligosaccharides. These oligosaccharides copurify with DNA since they can interact with it nonspecifically. I think they are able to interfere with some enzymatic reactions. We notice that gel purified PCR fragments cut less efficiently then those purified by PCR kit. Perhaps, these oligosaccharides can also affect spectrophotometric properties of DNA. Isopropanol othen helps components in mixture to precipitate with DNA, especially at low temperatures. Therefore, it is highly likely that oligosaccharides have precipitated with your DNA. To precipitate nucleic acids, we use the combination of ethanol (3 volumes) and sodium acetate (3M pH 5.2 take 1/10 volume of DNA probe).
They are right, spectrophotometry (includes Nanodrop) simply cannot distinguish DNA from RNA, as mentioned by Jan. At this point, we cannot tell if your sample is really contaiminated with RNA. Run a low percent (~0.8 %, 150v ~15min for mini-gel) gel to check whether your sample is contaminated with RNA (with the presence of 28s 18s RNA bands). Good luck!
It may be several ways, either due to carry over or cross contamination by RNA of your sample or your DNA extraction or purification kit was previously contaminated. Even when you extract your DNA, then already it is contaminated. Always use a dummy to check your kit while extract DNA from the sample as a negative control, play just without sample and run what happens.
You already have great answers advanced. Depending on the Nanodrop you are using, make sure your setting is for DNA not RNA. If you leave it at the RNA setting, you should still have a reading which might cause the confusion you are having. However, since you have the expected DNA size on your gel, I would proceed with my experiments. If you want to be sure you have no contamination in your purified DNA, its better you pass it through a DNA clean and concentrate column once again. Good luck
i think the nanodrop just calculates concentration for DNA or RNA based on what its set on.You are pirifying a PCR product. Where would the RNA have come from?
Yes, UV spectrophotometry cannot distinguish between RNA and DNA as mentioned by others above. The chromophores are the same. If you suspect RNA contamination try adding a small amount of RNase to an aliquot. Leave another aliquot untreated. Incubate both aliquots at room temperature for ~30 min. Then, run both samples on an agarose gel 1-2%. Do you see a difference between them? If there was RNA present you will see a difference. If there is no RNA present for the RNase to work on then there will be no difference.
You had good result with the gel, so estimate from it. If you are to use the Nanodrop, close the program and restart it. Set it to read as DNA not RNA, and repeat the process twice. Overall, rely more on your gel result.
Thanks everyone. I appreciate the advice and answers and will implement this new knowledge as I continue the research. Thank you all very much
Since it is DNA that gets amplified in PCR and not RNA obviously the nanordrop is unable to distinguish DNA from RNA. Nanodrop can only detect the total nucleic acid present and DNA and RNA separately
Sivaramakrishanan is right. You amplified DNA by PCR not RNA, then nanodrop is unable to distinguish DNA from RNA.
You can treat your reaction product with RNase and make sure that you have only DNA and then go by the right method
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