Plasmid DNA purification - DNA purification (Sep/03/2004 )
We are looking for a plasmid purification technique (kit or manual) that fulfills the following criteria:
1) begins with an approximate 50 mL culture
2) can prep 20 at a time, with or without vacuum, or spin
3) give DNA clean enough for electroporation
4) doesn't use phenol
We find that a typical alkaline lysis procedure from 1.5 mL culture of Miniprep DNA give about 40 ug of DNA; yet Qiagens plasmid preps (and Sigma which we have also tried) have given us less than 100 ug cumulatively when we do 50 mL preps. This means that column purifications are very inefficient. Can one scale up Maniatis efficiently, for example?
I use Qiagen's Maxi prep and always grow more volume than the suggestion for the kit. Also I find that using a beveled flask can give me twice as much DNA than using a regular flat-bottom erlenmeyer flask. On average for 300mL culture, the Maxi prep will give me about 600ug of 6.0 to 7.0kb plasmids as long as I use a beveled flask. Also make sure that your flask volume is 5 to 10x larger than your volume of culture; also depending on cell type and plasmid size you may have to optimize the incubation time, we found that certain types of cells would give better yields at just 12hours instead of 14 or 16hours. Columns are not terribly efficient, but do cut time, especially with the high speed kits. The only other option that I know of is to do CsCl preps which will take you a minimum of 2 days no matter the scale of the prep. Qiagen's giga prep will yield 8mg on average of good quality DNA but it does take just as long as CsCl preps, but it is much easier. If you are planning on electroporating mammalian cells though, I'm afraid the only way to fully clean DNA is to use phenol:CHCl3 extractions.
We've routinely used Epoch Biolabs' GenCatch Maxiprep Kit for ultra-clean plasmid DNA for transfection, in vitro DNA repair and in vivo vaccination...
The quality and yield (600-700 ug per 200-300 mL culture) are astonishing and very satisfying. Most importantly, the price is affordable and much less than Qiagen's. If you're interested, check their web site www.epochbiolabs.com
I have used a Maxi-prep from Bio-Rad.
It is working just fine, it fullfills the criteria, but i do not know
how many samples you can run at the same time.
Maybe not 20...
you can use the maxi prep method as quoted in mainiatis
We at Epicentre are launching a kit early this year (PlasmidMAX) that may fill the bill for you with mutiple plasmid preps, high yield. I just did a 50 ml scale prep of a 5 kb plasmid that gave a bit over 500 ug of "honest" DNA, measured fluorimetrically. It beat the pants off of the Qiagen FastPrep Midi kit. Stay tuned for details...
I wonder how u get 40ug of Plasmid DNA from 1.5 ml culture. I never got that much.
If u allow me I would like to use your protocol.
I work for Promega's Technical Services deprtment. The requiremtns you have meet the specifications of our new PureYield™ Plasmid Midiprep system. Witha vacuum manifold exactly 20 samples can be processed at one time from 50mL of culture and we typically see well over 200ug of DNA. Using the link beow will bring you to a survey that allows you to receive a free trial of the system.
If you have any additional questions, please feel free to contact me here, or preferably by e-mail at firstname.lastname@example.org