As we know, a 260/280 ratio of ~1.8 is accepted as pure for DNA. Recently, I extract my plasmids in order to transfect, but some of them are in higher or lower rates of 1.8 !!! (The lowest is 1.4 & The highest is 2.3). In your opinion,the plasmids with this ratios can reduce the transfection efficiency?
Thanks,
Technically speaking, plasmid concentration won't influence the "efficiency" of transfection - it's just that you'll be transfecting all of the extra debris with the plasmids as well. Having pure plasmids is of course important, but for efficiency you'll need cell line tailored reagents (like these: https://altogen.com/products-index/) and make sure to have control samples to see if you're getting good results for future protocols.
Dear Leila,
High purity DNA is important for successful transfection. The OD 260/280 ratio should be between 1.7–1.9. Higher or lower ratios indicate impurities and should not be used in transfection experiments. In order to overcome the problem, I suggest to get cleaner plasmid DNA for those samples with doubtful OD.
Good luck.
You are always safer using DNA that you know is clean and has a good ratio. But it depends on the nature of the contaminant as to whether or not your transfection will work adequately or not. One thing I would certainly do is to run your plasmid on a gel to verify its concentration given that your ratio is so far off, so its concentration might be as well.
Dear Michael
Thank you. The concentration of my plasmids are more than 150 ng/μl. Nevertheless, I will run them on a gel.
Kind Regards,
Remember that your concentration is probably done by measuring A260, which is suspect. So while that may be the concentration of nucleic acid in your prep, it may not be the concentration of plasmid. RNA, chromosomal DNA, sheared DNA can all contribute to the A260.
Dear Michael
Yes that's right. How can I make sure that there is no RNA or chromosomal DNA in my samples?
Dear Leila,
I agree with Michael. Production of plasmid DNA creates purification challenges related to removal of various contaminants such as chromosomal DNA, RNA, and endotoxins. These contaminants have similar properties to the target molecule. Therefore, strategies must be developed for their removal.
One of the techniques that can be used for carrying out such task is PlasmidSelect Xtra resin, based on thiophilic aromatic chromatography, which can separate plasmid from host contaminants and provides good resolution between open- and closed-circle forms of a given plasmid DNA.
Please find attached the paper of Hitchcock et al. (2010) for more details.
Best Wishes.
Dear Temim
Thank you very much for your good information. These can be helpful and excellent for me.
Best Wishes.
Technically speaking, plasmid concentration won't influence the "efficiency" of transfection - it's just that you'll be transfecting all of the extra debris with the plasmids as well. Having pure plasmids is of course important, but for efficiency you'll need cell line tailored reagents (like these: https://altogen.com/products-index/) and make sure to have control samples to see if you're getting good results for future protocols.
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