I need to extract large amounts of plasmid DNA for transfection into mammalian cell lines with as little as possible enterotoxins and I usually use the Qiagen Endofree plasmid maxi kit for the extraction. When doing miniprep with the Qiagen plasmid miniprep kit extractions to select colonies for the maxiprep I get excellent yields of over 300ng/ul with a 60ul elution. Since returning to the lab in 2021 I have tried a PureLink™ HiPure Plasmid DNA Purification Kit and the Qiagen Endofree plasmid maxi kit and got less plasmid out of these kits than from the miniprep kit.
Do you have a possible reason for the low yields and suggestions for me to improve the yields
Low yields of plasmid DNA can be caused by a number of different factors. The most common causes for low yield are poor culturing conditions and plasmid propagation, excessive amounts of starting material resulting in insufficient bacterial cell lysis and column overloading. When working with the anion-exchange based QIAGEN Plasmid Purification Kits, extra care is required during the isopropanol precipitation step, as the glassy DNA pellet may be difficult to see, and tends to be only loosely attached to the side of the tube.
We strongly recommend to review the information provided on our Plasmid Resource Page in the section 'Optimal results with QIAGEN plasmid kits', as it provides useful background information on growing bacterial cultures and general considerations for optimal results. It is also necessary to follow the instructions in the relevant protocols precisely to ensure the best plasmid yield and quality.
To determine at what stage of the procedure any problem occurred, save fractions from different steps of the purification procedure, and analyze by agarose gel electrophoresis. For a detailed description on how to run and interpret an analytical gel, please see Appendix A in the QIAGEN Plasmid Purification Handbook: "Agarose Gel Analysis of the Purification Procedure", or visit the QIAGEN Plasmid Resource Center.
This could depend on a lot of factors: growth phase of the culture, amount of starting material, etc.
When you say you got "less" plasmid with the maxi kits, do you mean an overall smaller amount (fewer micrograms) or do you mean a lower concentration? As long as you are getting enough to use, I wouldn't really worry about it.
What's your goal - to get a certain amount of plasmid (e.g. need X micrograms) or to get a certain concentration of plasmid (X nanogram/microliter)?
You can always change the final concentration by either evaporating or diluting.
Low yields of plasmid DNA can be caused by a number of different factors. The most common causes for low yield are poor culturing conditions and plasmid propagation, excessive amounts of starting material resulting in insufficient bacterial cell lysis and column overloading. When working with the anion-exchange based QIAGEN Plasmid Purification Kits, extra care is required during the isopropanol precipitation step, as the glassy DNA pellet may be difficult to see, and tends to be only loosely attached to the side of the tube.
We strongly recommend to review the information provided on our Plasmid Resource Page in the section 'Optimal results with QIAGEN plasmid kits', as it provides useful background information on growing bacterial cultures and general considerations for optimal results. It is also necessary to follow the instructions in the relevant protocols precisely to ensure the best plasmid yield and quality.
To determine at what stage of the procedure any problem occurred, save fractions from different steps of the purification procedure, and analyze by agarose gel electrophoresis. For a detailed description on how to run and interpret an analytical gel, please see Appendix A in the QIAGEN Plasmid Purification Handbook: "Agarose Gel Analysis of the Purification Procedure", or visit the QIAGEN Plasmid Resource Center.
Jaba Tkemaladze
The culturing for both the miniprep and the maxiprep was done the same except for the larger scale in the case of the maxiprep. With regards to the pellet after the isopropanol precipitation, the pellet was clearly visible and was firmly attached to the tube.
I will save fractions during the different steps in the future to help with the trouble shooting.
Katie A Burnette
When saying I got less plasmid I am referring to the overall yield of the plasmid.
The culturing of the bacteria was done in the same manner just on a large scale for the maxipreps.
I am going to use the plasmids for transfection in a reverse genetics system and would prefer as much as possible plasmid from a singe extraction as to remove extra variables during the various experiments in the rescuing of my viruses.
Look, the goal of the plasmid prep is to create plasmid as a reagent for your experiments.
Don't waste time trying to get the absolute highest yield (saving fractions, etc.). Go for "good enough is good enough" so you can continue with your actual project. If you can get enough plasmid from the mini preps, then just do the mini prep.
Also, remember to freeze aliquots of your plasmid so that you can avoid the degradation from multiple freeze-thaw cycles.
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