I extracted plasmid DNA (less than 7000 bp) through a miniprep kit and confirmed the amount of DNA in a spectophotometer. I then tried to digest the plasmids to confirm that my insert has been inserted in the plasmid vector.
It happened that my samples get stuck in the wells while the ladder runs normally (1st run). Undigested samples did not run in the gel as well.
I then performed another digestion reaction (with another restriction enzyme and buffer) and got the same result (2nd run, picture).
I used ~400-600 ng of DNA in the digestion reactions.
Previously I was able to digest and visualize in the gel ~500 ng of plasmid (from another cloning/miniprep batch), so the amount of DNA is probably not the issue.
Between the 1st and 2nd runs I performed a run of a PCR product (same loading dye as 2nd run, same TAE buffer 1X, 1% agarose gel and same equipment) and samples did run normally.
Answers to similar questions in Research Gate seem to point to large genomic DNA as the cause. But is it possible that contaminant genomic DNA from some source could bind my digested plasmids and stop them from running in the gel?
Does all of this indicate that the problem is in my minipreped sample or is there another possible reason for my problem?
Not everything that absorbs at 260 nm is nucleic acid and I strongly doubt that what you loaded on your gel was DNA, much less the expected plasmid. The stained material stuck in the wells is probably some insoluble garbage that binds ethidium bromide. No need to come up with fancy scenarios.
I agree with Pierre Béguin that the stuff stuck in the well is probably debris although it could be denatured genomic DNA. Regardless of what it is though, you clearly don't have a good yield of plasmid. It is also telling that I see no residual RNA debris that is frequently seen with plasmid minipreps. I would suggest troubleshooting your plasmid prep protocol and reagents.
Molecular junks will not enter the gel cos they are too heavy. It is likely you don't have plasmid in there or something went wrong with your methods.
Thanks everyone for the answers. In fact I got really bizarre ratios in the spectophotometer quantitations so I was iniatially worried... But then I was told that they didn't matter for plasmid preps. Seems like they do matter after all and that I had no purified plasmid to begin with.
After some troubleshooting I think I've found out what went wrong in my procedures. Thanks everyone again for the comments.
Missing a single step in the DNA extraction process could mitigate the product of the plasmid yields. A lot of factors tends to come into play. Be very careful while Re-running this process next time. Best.
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