Plasmid Purification problems

3 posts / 0 new
Last post
jseibert
jseibert's picture
Plasmid Purification problems

Hi all.  I am having problems with a step in the Wizard Plus SV Minipreps system for isolating plasmids by Promega.  After cell lysis ( as well as protease inhibitor and subsequent neutralization steps), you have to spin down the cell lysis solution in order to separate gDNA precipitate from the plasmid (I am getting good lysis as noted by the "stringy/sticky" appearance).  After I have spun down the tubes, I get a great pellet, but when I pipet off the supernatant I still see "debris" getting sucked up into my pipetman tip (although I am not disturbing the main pellet at the bottom).  I have tried adding 5 more minutes to the centrifugation step and have also added a second centrifugation step to try and pellet the stuff that I manage to suck up in pipetman.  Should I spin longer to get the other debris/floaties into the pellet?  Any ideas/suggestions would be greatly appreciated!

Jason King
Jason King's picture
 It is incomplete lysis and

 It is incomplete lysis and neutralisation. This could be caused by too much biomass (bacterial culture) being used to start with.

After addition of lysis buffer you should see gloopiness. Make sure that you carefully invert mix the tube and give enough time for full lysis. Then add the neutralisation buffer and again mix carefully via invention. Again, allow full neutralisation. You know it's full if the gloopiness vanishes. Then and only then centrifuge down. 

Most people use too much overnight culture for the prep and this makes the denat/neut steps less efficient. It also makes binging of plasmid DNA to the column less efficient. Especially if you are using a high copy plasmid - LESS IS MORE.

if you have a low copy plasmid I have another trick but try the above first.

Good luck,

PM

jseibert
jseibert's picture
Thanks, much appreciated!!

Thanks, much appreciated!! Come to think of it I probably have been using too much (10 mL o/n culture)  so I will give this a try and let ya know how it goes.  Thanks again.