Anyone who has worked with recombinant DNA has most likely done a maxiprep and when it comes to commercially available kits, there are several to choose from these days. Qiagen products are a staple in our lab, from DNA plasmid preps (mini, midi and maxi) to gel extraction kits. We use Qiagen’s Plasmid Maxi kits to isolate large quantities (500-800 ug) of DNA suitable for sequencing, restriction digests and even for transfection of mammalian cells. We continue to use these kits because they are reliable and give excellent yields of pure plasmid DNA (good 260/280 ratios).
The basic protocol is similar to most maxiprep kits. The bacteria is harvested by centrifugation, resuspended in buffer, lysed and neutralized with buffers included with the kit. After incubating on ice, the sample is again centrifuged to remove the precipitate and the lysate is loaded onto the provided column. After all the lysate has passed through the column, it is washed twice and the DNA is eluted. Isopropanol is then used to precipitate the DNA (very important to thoroughly mix the sample to ensure the DNA precipitates) and the sample is centrifuged again. Depending on the yield, a decent sized white pellet is observed at the bottom of the tube. The pellet is then washed, dried and resuspended. It should be noted that the newer kits have a visual lysis control to help ensure the sample is completely mixed after lysis and neutralization; however, I have not tried this feature myself. Compared to other Qiagen kits, I personally prefer this kit over the QIAfilter Maxiprep Kit (after lysis and neutralization, lysate is cleared through a syringe instead of by centrifugation) as I seem to get a higher yield of DNA. I am not sure if this is due to losing DNA in the filter or because it is difficult to get all of the lysate through the syringe.
We have modified the protocol over the years in an attempt to increase yield and reduce the time it takes to complete the maxiprep. We often grow twice as much bacteria (200 ml) and use twice the volume of the buffers (20 ml) to resuspend, lyse and neutralize the sample. The larger volume is then loaded onto one column (done in steps) and the rest of the protocol is followed as normal. With the increased amount of bacteria, we often get 700-800 ug of high copy plasmid DNA from one maxiprep. The only problem with this alteration is that it requires increased volumes of the buffers. To get around this, we purchase the additional buffers and RNaseA from Qiagen, but the buffers could be made in the lab as well (compositions are included in the manual). It should be noted that this alteration does not work with the QIAfilter Maxi Kit, as the syringe is not designed to hold the increased volume.
To save time, instead of centrifuging the sample twice to clear the lysate (following lysis and neutralization), we only centrifuge the sample once and pass it through a kimwipe (placed into a small funnel) before it is loaded onto the column. We simply fold the kimwipe to make a triangle and repeat to make a little cup. This method works very well to remove any additional precipitate that did not pellet during the spin and does not seem to affect DNA yield or purity.
Finally, after the 70% ethanol wash, we transfer the DNA pellet to an Eppendorf tube before drying. To do so, we cut the tip off of a P1000 tip (using a clean razor blade) and use 70% ethanol to transfer the pellet into the tube. We then spin 5 minutes at max speed in a desktop centrifuge and remove this final wash with a pipette tip. It usually takes two spins to remove the majority of solution before drying the pellet. We do this for two reasons: first, it ensures that the entire DNA pellet is in the Eppendorf tube before drying. This means you do not have to worry about whether or not the DNA has completely dissolved before you can transfer it to an Eppendorf tube. Second, if the DNA is going to be used for transfection, this allows us to sterilize the DNA (using 70% ethanol) in the Eppendorf tube. For this, we remove the final wash with sterile pipette tips and dry the DNA pellet in a sterile hood. The DNA is then resuspended in autoclaved water or filtered sterilized 10 mM Tris pH 8.5 (we often use Buffer EB from other Qiagen kits). I have found that the DNA dissolves better in the Tris solution than it does in plain water. DNA prepared in this manner has worked well for transfection of mammalian cell lines as well as for other molecular biology techniques.
These kits do have some minor disadvantages that should be noted. First, with all of the centrifugation steps and filtering through the column, the protocol takes well over three hours to complete. Although most of this time is not hands on and the column can be left for extended periods of time without drying out, it can still take half a day or so. Second, for some reason, we often get a small white precipitate when the dissolved DNA is centrifuged in an Eppendorf tube. I am not sure if this is undissolved DNA or some sort of salt precipitate that does not go into solution. However, it does not seem to affect any downstream applications. If it is a problem, the DNA can always be transferred to a new tube. Finally, these kits are slightly more expensive than some of the other kits available on the market. I have not done a direct price comparison as I haven’t found a good reason to switch to a different kit. With Qiagen’s Plasmid Maxi Kit, we get reliable results with good yields of pure plasmid DNA.