Not totally familiar with this kit but since most of Maxiprep kits work on the same principle I can try to point out one possible mistake.

After filtrating your Lysate/Precipitate on the syringe you have to add a binding buffer to your filtrate before to apply it on the DNA binding column. Did you do this step ? Omitting it results in very poor binding of the plasmid DNA to the silica column and ends up with super low amount of purified DNA

Maxiprep columns are actually anionic exchange, so the principle is different than in case of minipreps (silica columns). Due to that buffers used for mini and maxipreps are not interchangeable.

Wesley, what are the total yields of your plasmids? What are their copy numbers? Also, have you compared your DNA yields after column and after isopropanol precipitation? In case of maxiprep yields, I am always vary of the precipitation step, because the efficiency will depend on your starting DNA concentrations.

Ana Crnkovic I am unsure of the exact copy number of the plasmids, but concentrations were 0.1 ng/uL and 89.2 ng/uL, obviously far under expectations for even low copy plasmids.

As far as differences in buffers between mini and maxi preps, I did use some buffers from a miniprep kit (R3 resuspension and L7 lysis) but these buffers have the same letter/number designation as those from a maxiprep kit with no discernible difference. Would these buffers still be incorrect?

I agree that it is unlikely the kit components have gone bad merely a few months after expiration date. I would suggest you try again and save a little bit of sample from before the column and also the subsequent washes etc and run on a gel. You need to determine whether you are not getting plasmid DNA at all or whether you are losing it during the column step. That way you will know what step to troubleshoot.

Wesley Hanson It should not be the resuspension buffer (usually just 50 mM Tris pH8 in all kits), nor the lysis buffer (usually 200 mM NaOH, 1% w/v SDS), but the neutralization buffer. I don't know exact composition of the kits you are using but with Qiagen Maxi you would neutralize with 3.0 M potassium acetate pH 5.5, and in case of their minipreps with lower concentration of KOAc and at lower pH; you would also have GuHCl here. So after you neutralize and pellet the sample, your supernatant has the optimal pH and salt concentration to be immobilized on either anionic exchange resin (Maxi) or silica columns (mini).

I could not find the exact composition of the buffers you are using, but I highly doubt that R3 or L7 would influence the outcome/yields. My suggestion is to collect DNA after each step (supernatant after you neutralize, FT after binding to the column, all of the washes, elution and then the pellet after isopropanol precipitation). It's the quickest way to determine what went wrong.

Good luck!

Is one of your reagents NaOH or KOH?

These solutions tend to absorb CO2 from the air to form bicarbonates.

I remember from the days of doing Birnboim-Doly preps (the ancestor of many modern kits) that if the alkaline-SDS solution was not fresh the plasmid preps were poor.

Andrew Jenkins I've also been having trouble with invitrogen HiPure with the syringe precipitator module. I get no DNA every time no matter the plasmid. I have miniprepped before and can confirm there is plasmid there in the culture. I also have tried a different company kit and can purify the plasmid fine. I can see there is DNA in the flow-through after isoprop and ethanol steps. Seems there is something wrong with the precipitator module filter. I'm switching to a different company now but trying to figure our the best.

Wesley Hanson Qiagen has started manufacturing Qia Plasmid plus midiprep kit. The volume required is as low as 25 ml and my own experience has lead to yield 1.2 ng per micro litre for a kan resistance high copy number plasmid. In between can you tell what is the copy no. of the plasmid. Andrew Jenkins has rightly pointed out fresh P2 for plasmid extraction is good. Also in amp resistance plasmid, there is a perpetual problem of loss of plasmid as sometimes the strains overexpress betalactamases.