Hi,
I extracted the plasmid manually and measured a concentration of 12,000 using the Nanodrop. However, nothing shows up on the gel electrophoresis.
Are there any specific techniques or tips I should follow during the extraction process? I’d really appreciate your guidance.
Thanks a lot🙏🏻
You have a very strong band at a low molecular weight which is either RNA or degraded DNA. So when you measure with the nano drop it is only measuring nucleic acid so all the stuff at the bottom is giving you a high measurement.
If you treated with RNAse during your plasmid prep then probably it is degraded DNA. If you did not treat with RNase then it most likely is just RNA.
What size is that DNA ladder and what size is your plasmid?
Whatever you have on that gel is "screaming bright" - that means there is a lot of it. And the extreme bend/smile in the bands means it's too much for the well & it didn't run true to size.
Try loading just 1 microliter and see if you can get better resolution to at least know a size estimate.
I agree with Michael J. Benedik and Katie A S Burnette; it looks like there is something else in your gel that is not intact plasmid and is producing a strong signal. Moreover, a NanoDrop reading of 12,000 ng/µL (I assume those are the units) is likely too high to be reliable.
To give you a more specific answer, I'd need to know:
- The scale of the plasmid preparation (mini, midi, maxi, giga).
- The bacterial strain employed.
- The specific growth conditions (medium, temperature, duration).
- Whether an RNase treatment step was included.
Well you do have a band! Meaning that the DNA is there! But not in one piece! To my experience, you are losing your plasmid as a result of degradation. I recommend using a commercial kit instead as the quality of plasmid is pretty important in downstream tests anyways.
Well, something is showing up on your gel. It's some kind of DNA and/or RNA as it's binding enough of your gel stain to leave behind an empty "shadow" of no stain. What size is your plasmid and what size is your ladder?
I'd suggest diluting your sample, redoing the nanodrop, and rerunning the gel so you can get a more informative picture.
Ok, when you say the nanodrop gives a reading of 12,000, what units? Ours does ng/ul, are you converting to a different unit? I don't think ours even reads units that large.
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