Hi, I'm a master's student in biotechnology and I'm having trouble visualizing plasmid bands during extraction. I'm working with the plasmid pET-32a in *E. coli* DH5α. I've tried both kit-based and manual extraction methods, but I can't see any plasmid bands. I've run the electrophoresis at 90 volts for 40 to 45 minutes (1% agarose gel) using Safe stain for staining. After using the kit extraction method, the Nanodrop showed a concentration of 162 ng/µL. The DNA ladder shows clear and distinct bands, but the plasmid does not. Does anyone have suggestions on what might be going wrong or how to improve the visualization of plasmid bands?
Dear Bahar Hatefi , welcome to the realities of research. So there could be several reasons you don't see your plasmid on the gel. The most probable reason is that your extraction process did not lead to the elution of the plasmid in the end. Now this is not certain since you have a concentration on NanoDrop. But one other possible reason may be that the quantity of plasmid is too low. So I have some questions as you troubleshoot:
1. Did you clean the NanoDrop before using it?
2. What did you use in blanking the NanoDrop?
3. How much DNA did you load on the gel?
Dear Robert Adamu Shey , Thank you for your response and guidance. To address your questions:
1. Regarding the NanoDrop, I did not perform the measurements myself, but my colleague did. I will confirm with her if the instrument was cleaned properly beforehand.
2. For blanking the NanoDrop, I believe nuclease-free water was used. I will double-check this as well.
3. On the gel, I loaded 1 µL of plasmid DNA and 3 µL of 6X loading dye.
Do you think the issue might be related to the loading volume or perhaps the amount of DNA? Would increasing the DNA sample or changing the loading conditions help improve visibility?
Thank you again for your time and suggestions.
From the gel picture it seems very clear that you have no plasmid DNA. There are two possibilities here, the first is that there is no plasmid in your prep and the second would be that the DNA went away during digestions. If you load some undigested plasmid on a gel to see if there is anything there that would tell you the which it is. Given that there is no visible RNA or sheared DNA, I think it most likely that there is nothing in your plasmid prep.
Nanodrop only tells you that something is absorbing, not that it is any good. If you had DNA or RNA that was all degraded it would still give you a high Nanodrop reading. Also chemical contaminants do the same.
The question to ask the person who ran the machine is what the curve looked like, is it a nice curve with a peak at 260 or did she just give you the 260nm reading?
But the bottom line is that a gel picture is more accurate than the nano drop.
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