Hi, it should work. I have isolated plasmids about 40kb by standart miniprep kit mady by invitrogen. And since the mechanism of these kits is identical and quiagen is a good company...it should work. my opinion is that the smaller band you can see is the supercoiled form of your plasmid---and since it is supercoiled, it travels faster and looks like smaller molecule. If you have same molecule, say 20kb: linear form will be slowest, then would be circular form and then supercoiled (fastest). Same molecule size have different mobility on the gel accordinbg to its conformation. btw, you can not determine size of circular molecules according to linear size marker. These circles will look smaller than they actually are. I hope it hepls.

The Qiagen prep works for plasmids up to 80-90 kb or even larger. The problem is that around 40 kb, the elution becomes more problematic so the yield starts dropping. Plus shearing becomes very obvious. Do note that heating nicks your DNA. I never heat up more than 50C. Heating at 65C is not recommended. Some sources advise to get DNA pellets into solution by putting at 65C. Yes, that works, but it also nicks. If your DNA needs to be integer (for transformations, transgenics), this is a problem. Better then is to leave at 4C overnight, spin and transfer supernatans to new tube.

Also, the migrations of the DNA forms changes a bit. For small plasmids you have supercoiled (fast), linear, open cicrular (slow). At some point, I would say around 25-30kb, the supercoiled runs equally with the linear. Above that size, linear (fast) will always run at the same height (no matter what size of plasmid), then superocoiled and open circular (slow). Some of the DNA will get stuck in the well too depending on eletrophoresis conditions. The larger the DNA, the more DNA gets stuck in the well. The supercoiled will run at a height relative to the size of the plasmid. That is why you can "size" large plasmids on a regular mini gel based on supercoiled and not linear; for this you need to use a supercoiled ladder (Epicentre) and run along a regular ladder. This method works for extreme plasmids like BACs.

In your case, the small band is strange. I do not think this is a true band, since it is not there with the larger preps. Maybe this is because you heat the sample extensively, I am not sure. Perhaps you can try a mini version of the drip column. Qiagen and Purelink sell mini prep versions of the drip/gravity columns that work identical to the midi/maxi prep. They take a little longer to perform compared to the centrifugation columns, but not as much as the midi/maxi, and they are kind of affordable.

Hi,

Column methods are not suitable for large plasmids as large plasmid are prone to breakage. I was working on Cupriavidus metallidurans CH34, which carries two mega-plasmids of 180Kb and 240Kb and the column methods were not suitable. I had great luck with this phenol protocol as per this paper "Rapid procedure for detection and isolation of large and small plasmids",C I Kado and S T Liu. Phenol has several advantages over column methods to get a high-quality plasmid as they will precipitate everything except circular plasmids. Gud luck, Senthil

What you see is the plasmid in its two forms ... The coiled and the super coiled... The super coil is sometimes/if not always ....become resistable to be digested with restriction enzymes...but the larger sized form is ok ...

I am agree with Senthil Kumar. I used this phenol protocol several times for isolating big plasmids and it was successful.

Hi all, thanks for all great comments ! Is there any possibility for oligomerization of plasmid or insert?