| | Re: Very low plasmid yields from mini-prep
For the Qiagen spin miniprep kit, here are a few things that I've found helps yield.
1) Don't use too much O/N culture. Although I've never run a side-by-side comparison, I tend to get better yields using 5-6 mL rather than 7 or 8. More is not always better!
2) If your kit has LyseBlue, use it. When I started adding this reagent to my preps, I noticed that my usual way of handling samples didn't always make the color turn the way it should. To be precise, I tended to mix too few times after the neutralization buffer addition step.
3) Load your sample through the column twice. This may be my own little paranoia, but I like to run the lysate through twice (take what went through the column into the collection tube and reload it into the column) and the elution through twice. I don't think it hurts anything, and I figure it gives molecules one more chance to bind (or elute).
4) Let the wash evaporate completely. I've noticed, even spinning an extra minute to get rid of the wash buffer, I can often still smell the ethanol. Now I like to let the columns sit and air dry in the elution tubes for a couple of minutes before I add the elution buffer.
5) Heat the elution buffer. One I've never tried, but it's suggested in the protocol (I think) -- Heat the elution buffer to 65 degrees before adding it to the column (and I might incubate the column with elution buffer in a heat block for a minute as well) -- the heated buffer is supposed to be better at eluting DNA.
6) Don't throw away the column. I hold onto the columns while I spec my first elutions -- if a prep has a low concentration, I run another 50-100 uL elution buffer through the column and check its concentration. If both elutions have some small amount of DNA, I pool them, ethanol precipate, and resuspend in a smaller volume (say 10 or 30 uL). This is some extra time, but I think it's easier than trying to redo the entire miniprep.
Remember that yield for these kits is based on two factors: how much plasmid you put in at the beginning, and how much makes it through the purification process. I've talked about the second factor, here are some pointers on the first:
1) Check to see whether the plasmids that aren't prepping well are low copy number.
2) Keep an eye on the size of your bacterial pellet. Maybe you don't have as many bacteria to begin with.
3) Associated with both factors: I seem to see an inverse correlation between the size of the plasmid I'm purifying and the yield: I think larger plasmids (>10 kb) don't make it through the purification process quite as well (perhaps if they get nicked they get caught up with genomic DNA during the lysate clearing step? and/or they don't elute as well from the column due to their increased interaction area?), and I suspect they will have fewer copies per bacterium as well (though I don't have hard data on that).
Well, that's my $.02 (or was it more like $.25?)...