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Is that a Plasmid??? - (Feb/15/2012 )

Hi i recently ran a plasmid isolation experiment (pUC 18) using the standard alkaline lysis method...i did not use(skipped) the lysozyme or phenol-chloroform step...the gel is agarose...i have attached a picture of the gel..only 6 wells have been loaded, DNA has been precipitated in the 1st,3rd and 5th loaded well using ethanol and the remaining 3 using isopropanol.......i cannot identify what the smear is at the bottom of the gel and the bands present in the smear...also i wanted to confirm that the bands above are plasmid dna...any help would be appreciated.....thank you...Attached Image

-james castel-

I think DNA preps contaminated with RNA would run with a smear.


Unless you added RNAse to your prep, then I would assume this is large amounts of RNA. You can verify the plasmids if you can singly cut them with a restriction enzyme. The result should be a single band, rather than the mixed set of supercoiled, nicked, and circular DNA you see on your gel.


May I ask how you can tell they are RNA, rather than fragments of DNA? Sorry for this naive question. I am very curious! Thanks


Technically you can't. However, RNA tends to be small fragments of nucleotide between 20 bp and about 2000 bp (well, the visible bits anyway), and will co-precipitate with the plasmid DNA. Genomic DNA tends to run as a large band that may or may not just leave the wells at the top of the gel.


Thanks! Appreciate your answer~