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I am currently trying to see if 2 bacteria contains plasmids or not. I had used promega plasmid extraction kit on the bacteria. I ran a gel electrophoresis (.8% gel) with the extract product, however its not showing the presents of any DNA. I also ran the product through Nanodrop (2000) and these where the concentrations I got:

1 : 6.5 ng/ul & 260/280 = 1.56
2 : 49.4 ng/ul & 260/280: 1.96

Based on experience, I want to say #1 contains no plasmids, however #2 may have a small concentration of plasmids. Please give me your opinion on what to conclude. Also, if there is another test I can try to do to confirm plasmid presents or not, please let me know.

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    $\begingroup$ If your samples are contaminated with proteins (which can happen), then the resulting OD 260/280 gets higher and can falsely show some nucleic acids. If you run a few microliters on a gel and don't see anything, I would be suspicious. The concentration is sufficient to see something. $\endgroup$ – Chris Aug 19 '16 at 19:17
  • $\begingroup$ The amount of DNA is what matters when running the gel, not just the concentration. What volume of each sample did you run on the gel? Use the concentrations determined above to see how much DNA should have been there. If the hypothetical amount looks good, and you don't see anything on gel, I'd say there isn't any real DNA. As far as testing for presence of plasmid, you could try re-transfecting your suspicious sample into other bacteria, since you don't need many copies of the plasmid to get some good colonies. $\endgroup$ – user137 Sep 19 '16 at 1:43
  • $\begingroup$ This could be. I believe that the nano drop shows protein contamination. I am going to try to concentrate the plasmid DNA, and increase volume for gel. If I can not find plasmids, then I am going to assume that there are no proteins. $\endgroup$ – Adam Radek Martinez Sep 20 '16 at 14:13
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I don't know how much culture volume you used, but the amounts you have look more like a medium/high copy plasmid. I don't know if that's what you'd expect for a natural plasmid.

In addition to the other answer, it could also be sheared genomic DNA, cosmid (that would run too high on your gel to see), or maybe just nucleotides (if you messed up the prep in some way).

Another strategy to figure out if your strain has plasmids depends on what else you know about these strains. If you don't know that much: do 16S sequencing, it might be a known strain with known plasmids. If you already know it's a newly discovered strain: sequence the whole thing. Even with relatively low coverage the presence of plasmids will be quite clear. It's a bit more expensive than a plasmid prep + gel, but you'll also get a lot of other information about the organism.

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Having a positive reading on the Nanodrop but no band on the gel can be due to a number of different issues (not exhaustive):

  1. An incorrectly zeroed sample
  2. Incomplete staining of the gel
  3. Contamination of the sample
  4. Human errors in loading of samples

You should try to exclude these possibilities by positive controls in order to find out the true reason behind the inconsistent results.

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I would recommend switching up your PCR cloning program. Often, even after purifying DNA and RNA in conjunction with great care PCR can be the bottle neck for Gel Electrophoresis, as well as ensuring the quality (depth, void of blunt edges, etc) of the agarose gel- 49 ng/ul is not enough for progression. If all that still doesn't improve your case then it may be an issue with cultivating bacterica cultures -after purifying plasmids from bacterial clones vials should appear very cloudy (8-16 hrs was my go to time period), futhermore make sure to avoid light exposure on to the agarose gel as well as keeping it clean and submerged under cold PBS before inserting your buffered DNA (I used 10 microliters - but it tends to vary depending on the thickness of your gel) Also, when I would use a higher voltage during gel EP I would have a higher chance of having vertical streaking though it may be faster.

If nothing works... then I can only allude to possible human errors. Last thing! I hope you clean the nanodrop before you take count, having protein in your product can be a result of that. good luck! bacterica need love too!

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    $\begingroup$ Christopher - if you could, please add some references or external citations. We always appreciate it on this site. Thanks for your contributions! $\endgroup$ – Vance L Albaugh Sep 21 '16 at 11:11
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Depending on how you followed the protocol you could theoretical also have bacterial genomic DNA in your sample. If you follow the protocol strictly this should not happen, but if so, you would be able to measure the genomic DNA. In the agarose gel, however, it would usually be stuck around the top since it is too large to migrate.

Vortexing the sample before loading the column could cause this, for example (see Promega PureYield Manual).

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    $\begingroup$ Geo - if you could, please add some references/citations to your answer - I know at times it may be hard to reference something that may be common knowledge to you, but we appreciate it very much on this site. Thanks for your contribution! $\endgroup$ – Vance L Albaugh Sep 21 '16 at 11:09

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