5 replies to this topic

[james castel]

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...

Edited by james castel, 15 February 2012 - 08:40 AM.

[scolix]

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

[phage434]

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.

[CAT]

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

[bob1]

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.

[CAT]

Thanks!  Appreciate your answer~