After I did the mini prep, the concentration is about 300ng/ul and A260/280 is 2.13. However, no plasmid can be seen after I loaded 5ul to run a gel and there is a what I believe a RNA contamination showed below 75bps. I was wondering where does this RNA contamination comes from, i.e. from bacterial or from something else?
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It would be from the bacterial source. Perhaps you used a lot of cells to extract plasmid.
Hi Ali Javadmanesh
Thanks for the answer! But why sometime bacteria will give out RNA and sometime don't when I followed the exact protocol that gave positive results before.
If you are doing plasmid mini prep, are the reagents and consumables RNase-free? If not, I would suspect the low small base-pair fragment came from fragmented bacterial genomic DNA instead of RNA (which is highly susceptible to degradation by RNases).
Also, for your product concentration of 300ng/uL, if you multiply that by the volume you had, would it be far beyond what the mini prep kit states in the user manual about the yield? If that is the case, maybe what you got may be from the bacterial genomic DNA.
A possible trouble shooting solution: if you know the universal primer pair on your constructed plasmid, you can use your mini prep product as the template and the universal primers to amplify the sequence between the pair of primers on the plasmid. Then run the gel and see if you can see a band corresponding to the length between the primers. If yes, then you have your desired plasmid in your mini prep product; if not, then your template does not contain your plasmid, which might need you to get back to the step of transformation (maybe even prior steps) and check if the plasmid has indeed been introduced to the competent cells (Sometimes bacteria can still grow in the antibiotics-containing medium).