Concentrating plasmid DNA - (Sep/02/2005 )
I am having a problem concentrating plasmid DNA from large scale preps. The starting volume is 2L and final volume is 250ul in TE (pH 7.6). I ran an aliquot on an agarose gel after prep and plasmid is there. However, after concentration, I ran the same amount on a gel and see no plasmid. The method used for concentration was to add 1/4 vol 7.5M ammonium acetate and 2 volumes 100% ethanol and place at -80C for 1 hour. Then centrifuge at 4C for 30 minutes. A pellet is visible at this point. I washed the pellet in 70% ethanol and allowed to dry. This has been repeated more than once with the same results. What am I doing wrong?
Can you post your spec readings? Have you purified this plasmid before?
I think the amount of ethanol you are adding is too small. Normally one would use 2.5 to 3 volumes of ethanol, not 2 volumes. Your precipitate might be just salt (ammonium acetate). You didn't say what speed you were centrifuging at, either. I'd suggest a pretty high g spin, perhaps 8000-10000 g. Normally, I use 1/10th volume of 3M sodium acetate, pH 5.2 and 2.5 volumes of ice cold ethanol, followed by chilling at -80 until gelled, and a 20 minute spin at 10000 g. It is important that the mixture is well vortexed before chilling. Watch out for thin wall PCR tubes during this spin -- some of them will break. I'd suggest transfering to a thick wall tube. I rinse the pellet with 70% room temperature ethanol. You might consider using a co-precipitant such as yeast tRNA, glycogen, or a pigmented version such as Novagen pellet-paint. A co-precipitant is especially important if you have low concentrations of DNA in solution.
The final conc of Na Acetate used should be 0.3M.( I have 3M stock and I take 1/10th). Use pre chilled abs EtOH. Though 30' at -80 is enough try extending it up to 2 hrs or o/n at -20 also works.( I prefer iso propanol, 30' RT). Centrifuge at max possible. ( Beckman 13,000 rpm). Washing with prechilled 70% EtOh should be carefully done. I do not mix just add EtOH on opposite wall and spin further. You may lose DNA while resuspending, so mark the area where you see pellet when it is wet as it will disappear after drying. all the best!