I did my birnboim and doly prep and received multiple bands everytime. I compared the bands with samples using Qiagen miniprep kits and still I have multiple clean bands. When I added RNase to my Birnboim and doly prep the whole band just disappeared and there was a smear of white blob at the end of the gel. Am I doing anything wrong?
Hi,
Yes, multiple bands are normal in all plasmid preps. These are: supercoiled plasmid (covalently closed circular, CCC); relaxed plasmid (open circular, OC) and linear plasmid (L). In addition, some cloning vectors are prone to forming dimers in recombination-proficient cells.
The effects of RNase are rather startling, since they suggest that your bands are RNA, not DNA. The white blob (I assume that's just white on a monochrome photograph) would then be oligonucleotides resulting from degradation. I think it's actually more likely that your RNase is contaminated with DNase. That's easily checked by seeing whether it degrades a DNA size standard.
Are you running a DNA size standard alongside your plasmid preps? What are you staining your DNA with? Lastly, a picture would be worth a thousand words.
Hi Andrew, thanks for your suggestions. I did run a DNA size standard alongside. I stained my DNA with a coloured loading buffer. I've tried extracting my plasmid and insert using a commercial kit and the white blob seems to disappear. But I am still getting multiple bands. My colleagues suggested that my digestion did not work completely and that a part of my vectors are sill in coil form.
If you're still having problems, post a picture.
In the meantime, comparison with the size standard should show that at least one of your bands shows the migration expected of linear plasmid.
The 'blob', if it is ribosomal RNA, usually migrates well down the gel, corresponding to a DNA fragment less than 1 kb.
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