Altering Maxiprep procedure to prepicpitate with ethanol instead of isoproponal - (May/22/2007 )
I've been having a problem with my maxipreps. Everything goes smoothly until time for the final centrifuge and resuspension. I can NEVER see my pellet. When I add the isoproponal I can see the precipitation occuring but I can never see the pellet. I usually slowly decant and simply assume the pellet is still there. The one I did last night yielded a pathetic of 5 ug plasmid. My past ones have ranged from 20 ug to 300 ug. I'm working with pUC19 and incubate in LB 300 mL for 16 hours. Anyway, someone suggested I try precipitating with 100% ethanol instead of isoproponal. Firstly, why does the procedure even tell to use isoproponal, what is its benefit over ethanol? How would I alter the procedure to use ethanol? Simply add the same volume of ethanol I've been using isopropanal? Would the pellet be easier to see? Also, the procedure says to centrifuge immediately after adding the isoproponal, but should I incubate in a -20C freezer for some time first? Would it help to extend centrifugation time or go significantly above the Gforce suggested? At what temp. should I set the centrifuge (it suggests 4 C).
Sorry for all the questions but I want to get this right.
Thanks a bunch
If you would use Ethanol to precipitate, you will have to use 2x the vol of the DNA sol. unlike Isopropanol where you need to use only 0.7x the vol. of the DNA.
IF you could centrifuge at a higher g force after precipitating with isopropanol, then this might be a better idea to get a better pellet.
you don't need to chill the isopropanol after adding, and i think the ethanol ppt will be easier to see as there are more salts remaining so the EtOH ppt is more white while the isopropanol is very clear.
Another question: I got a suggestion to use glycogen to make the pellet more visible. How visible would this difference be and how much glycogen should I add?
pure DNA pellet is transparent , so you cant see any pellet if it is pure but still pellet is exist there, so just dissolve the dna in TE/H2O and check by running a gel
Novagen pellet paint NF at about 1 ul / ml of solution will make your pellet very visible. This is helpful, especially when you are first doing precipitations and have trouble finding the pellet. Another trick is to consistently orient the tubes, so that you know which side of the tube the pellet should be located on.
the big advantage of isopropanol over ethanol is the amoun needed. But despite the fact that general protocoles are talking about 0.8ml iproh for 1ml aqueous phase, i alway do in maxipreps extra isopropanol. I mean adding 25ml IprOH for 20ml aqueous for ex. chilling is not recommended for IprOH precipitation
You may use ethanol, but chill 30' at -80 would be good for yield. Also be sure using twice the volume.
For glycogen, i use 1µl pf 100mg/ml per ml of solution to precipitate.
Glycogen is solunble in ethanol as well as in water.
So 2-3 washes ensure to get rid of most of it