Problems w/ yield from lentiviral plasmid prep - (Apr/12/2007 )
Hello all, and hopefully someone can help me out. I am currently looking to use the ViraPower system from Invitrogen and want to clone my gene of interest into the expression plasmid. This plasmid is supposed to be transformed into Stbl3 cells (that's not a problem), but then I grow up cultures for mini/midi/maxi prep and get horrible results (~50ng/ul). I follow the protocol to a T and use the optional wash for endA+ strains (Sigma). Anyone else had this problem or found success your insight would be greatly appreciated. Thanks.
we use the same system from invitrogen. You have to grow a 10 ml culture for 8-10 hrs during the day and use it to start an overnight culture of 250ml. You should get around a 1mg of DNA.
You might also try using 2X LB broth to grow your cultures. That greatly improved my yield for the virapower plasmids. Also be sure to purify using the protocol for low copy plasmids.
Thanks to you both, I will try those ideas out.