Hi all,
I made different minipreps starting from the same sample of bacterial culture because my intention was to increase the concentration of plasmid in my final plasmid preparation. Now I have 5 tubes with 50 ul of plasmid each (the eluition was made in water). I was suggested to combine all these samples in one tube, put these 250 ul in a miniprep column, spin down, recover the column and eluite with 50 ul of water. In this way the sample should get concentrated from 250ul to 50 ul. Does it make sense for you?I had a doubt about this. If water is used for the eluition in the miniprep, don't I loose the plasmid when I spin the 250 ul that are in water in the column ?
Thank you
Silvia
You are correct, you would probably lose a significant portion of your plasmid using that strategy. Here are some alternate strategies that I'd recommend.
1. (This is similar to what was suggested to you) If you have a DNA gel extraction kit or a PCR purification kit, you can mix your plasmid solution with the buffer used to load DNA on the columns, then was and elute.
2. You can speedvac the DNA (easiest protocol in my opinion) assuming you have access to one
3. Mix the solution of plasmid with an equal volume of isopropanol (this precipitates DNA), then centrifuge at high speed for several minutes, remove the supernatant, allow the tube to air dry and resuspend the DNA in whatever volume of water you want.
I agree with James's recommendations especially no. 2&3 and I would also recommend lyophilization if you have access to a freeze dryer.
However, you can save yourself from this extra step through optimizing your miniprep and get a high concentration of plasmid at first hand. To do that, you can first pellet cells from a higher volume of culture (up to 5ml for high copy plamids and up to 10ml for low copy plasimds). Secondly, reduce the volume of the elution buffer to 20-30ul. Third, increase the incubation time of the elution step to 3 minutes at room temperature. Finally, be sure that the overnight culture always reaches the stationary phase before you conduct miniprep.
1. Yes, you will definitely lose some DNA. I have tried to find out this by adding 50 uL water to elute a column with DNA (get eluant 1). After spin, added another 50 uL water to the column and spin again (and collected the eluant 2). I continued doing the same procedures for 5 times and obtained eluants 1,2 3, 4 and 5. I ran all on a gel and found out all of them have significant DNA samples. (Note: 1. after adding 50 uL water, I let it sit 10-15 minutes, at room temp or at higher temp. 2. Have done several similar tests)
2. I like SpeedVac, as suggested earlier by James. The machine has 'cold' and 'warm' temperature setting while it spins. I usually used the 'cold' setting to concentrate my DNA.
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