I was given a tube of GFP DNA to use for a transfection and the quality of the DNA according to several repeats of readings on the NanoDrop is very poor. The A260/A280 ratio is around .6. Is it possible for me to run the isolated DNA through the isolation kit once more to clean the DNA and get a higher ratio? Or would that harm the DNA in the isolate?
I am using the QIAamp DNA Blood Mini Kit.
Hi Raven,
I have a suggestion to the problem. You can surely rerun through another column purification to improve the 260/280. However, with every purification step there is a loss of the DNA. You have not mentioned the A260 or the quantity. so first try to get the quantity of the DNA by A260 or agarose gel. If it is sufficient then go for another purification step. It wont harm the DNA except some losses.
Alternately, if your DNA is a plasmid, first retransform and grow it in bacteria, get sufficient amount of the pDNA, repurify it if required and go for the experiments.
best of luck
Sib
I also do not think it will help much, re-purification will reduce the amount of DNA.
Is it plasmid or genomic DNA? A kit for gDNA purification would not be the best product for cleanup of plasmid DNA, since binding conditions are optimized for large size fragments. I would rather take a plasmid or a cleanup kit (e.g. Qiaquick/MinElute) for such purposes.
Yes, you will loose some DNA, but are you really sure there is DNA in there? A ratio of .6 is very unusal, even if the the DNA is dirty it will seldomly have a ratio below 1.0. As suggested above, put an aliquot on gel, to get an idea of what's in there.
If there is DNA in there, but it has a low concentration (below 20 ng/µl), the Nanodrop is not a reliable instrument to measure ratios. To my experience the Trinean Xpose is much more reliable in this critical concentration range.
i think you will loss your DNA if you start extraction protocol from the 1st step, because there must be a many steps in protocol which will have DNA in pellet and other unwanted things in supernatant and at that position you will loss your DNA because you are not extracting it from the any cells, you just want to purify the extracted DNA so it would be good to not use that kit extraction protocol for purification.
You can precipitate the same DNA with double volume of chilled absolute ethanol and then by pellet the precipitates you can give two to tree wash with 70% ethanol. then you should check any good change in the ratio.
You should also check the DNA quality on agarose gel too. if gel shows a good quality and higher intensity of band then you can also extract highly pure DNA from that gel portion which having DNA band by different methods. but in that case you will loss your DNA a little by extracting from gel.
As jigar sutar said, you can precipitate the DNA with double volume of chilled ethanol following evaporation keeping the eppendorff open at RT and then redissolve the DNA in elution buffer (use less volume than you used previously). In a way DNA conc will increase so will increase the yield per microlitre.
Dear Raven Baxter-Christian,
The concentration will increase if you elute the DNA (from the silica column) by less elution buffer. Like, if 50 ul is mentioned in the protocol then you can give 30 ul, then the concentration and ratio will be improve a little bit.
Good luck !
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