Hello Tias, fine, very good... I couldn't believe there is still somebody working the good old way. I don't know what you loaded on gel, although it is indeed a lot, but it looks good. I think for easiness of handling, diluting the plasmid to 2 ug/ul as I was saying before will be better. It is difficult to have a uniform concentration when the DNA is so viscous, which means you will always take out different amounts depending where the tip of your tip ends up in the solution. And in general to evaluate the quality of the DNA I would load 200 and 500 ng on gel, you will get a much sharper run and you will be able to see better how much supercoiled and nicked form you have, as well if there are any concatenamers and so on, besides being able to confirm the concentration on gel.

Hi Tias, I doubt you really have that concentration of DNA, it would be so dense and viscous that you would not be able to pipette it, and you would need a very high copy number plasmid and an alkaline maxi-prep from 100 ml of culture to extract that amount (no column based midi or maxiprep will have that capacity!). Probably most of it is RNA debris, that's the problem with quantifying a midi/maxi-prep just by UV absorbance.

If you really want to know the concentration of your plasmid DNA you should load it on agarose gel. My guess is that the plasmid DNA is about 10 times less than you think. On agarose gel, supercoiled plasmid runs well when you load between 100 and 500 ng. So my suggestion is that you dilute your stock 1:10 in water or 10 mM Tris-HCl pH 7.4 and load 1 and 2 microl. If you have an appropriately quantified plasmid (form commercial source or from colleagues) you should load it in the same gel to help you determine the concentration of your plasmid, otherwise you can try loading a DNA marker with known concentration of the bands (for example 1kb plus from Invitrogen/ThermoFisher has 20 bands of about the same quantity (except two), so if you load 500 ng each band will be approximately 25 ng). Keep in mind that markers are linear bands and plasmids are circular molecules, whether supercoiled or nicked, and the run is different, as well as the appearance on gel, straight for the linear marker bands and smiling and/or roundish (depending on the amount you load) for the circular DNA.

In any case storing the plasmid up to 2 ug/ul will be fine.

Good luck.

Dear Boyl,

I know it is a very high concentration, but I usually and always get this amount (15-20 microgm) from 100 mL cultures. It is indeed very viscous and difficult to pipette.The vector backbone is pCDNA3, which is a high copy number plasmid. I do not use any kit based DNA isolation method. We do it manually, following the CSH Sambrook protocols. I agree that there is and will be some residual RNA even after LiCl precipitation and RNase treatment, but I doubt my quantifications to be wrong solely due to the presence of contaminating RNA.  I'm attaching the gel image for your reference.

Hello Tias, fine, very good... I couldn't believe there is still somebody working the good old way. I don't know what you loaded on gel, although it is indeed a lot, but it looks good. I think for easiness of handling, diluting the plasmid to 2 ug/ul as I was saying before will be better. It is difficult to have a uniform concentration when the DNA is so viscous, which means you will always take out different amounts depending where the tip of your tip ends up in the solution. And in general to evaluate the quality of the DNA I would load 200 and 500 ng on gel, you will get a much sharper run and you will be able to see better how much supercoiled and nicked form you have, as well if there are any concatenamers and so on, besides being able to confirm the concentration on gel.

Dear Boyl,

I usually dilute it to 1-4ug/uL and use it for transfection. I know that to observe all the 3 forms of DNA in gel, we should load low amount. I usually load 500-1ug. This was just a representative picture to show that indeed it was a very high concentrated DNA sample and not contaminated by RNA (even in the initial running phase, pic-maxiprep1, there is no RNA blotch seen running lower to DNA band). I wanted to know the disadvantages of storing it at such a high concentration. Thanks a lot for the clarification. I know  with the new methods and facilities very few or may be no one does it in the 'good old way'. But I prefer that, as the quality and quantity of DNA isolated is far better. The only pay off is time. But believe me its worth it!

Bhowmick,

I only meant for storage purposes. If storing it in a high concentration would affect its quality in a long term. I would dilute it for any downstream usage like PCR or transfection. Peitro rightly mentions that there would be pipetting error while making dilutions from the concentrated stock. But surely I won't ever be using such concentrated DNA for PCR. 

It seems to me that your question is the storage problem, well if it is so viscous and you never work with that concentrate DNA I dont see a problem if you diluted it to a much common concentration and of course storage making batches of it. This will save intact your DNA and free of contamination because you will use one storage sample for your experiments and contamination will be no issue, besides DNA will be better diluted and the amount you need will be perfectly measured.

Good advice above Pietro and Teresita. If you store your plasmid at 4º C, it will slowly evaporate over time and would get more viscous and concentrated. Diluting as above to a good practical concentration for your work (you could even re-OD the dilution to be certain) and then freezing aliquots and keeping a 4º C working tube would be a good solution. Then, you don't have to spend as much time diluting the DNA for each transfection and wondering about how accurate you were pipetting the viscous plasmid sample from experiment to experiment.