Just received pCFJ350 from Addgene and noted it’s in TOP10/P3 that carries the P3 plasmid (KanR, Amp (ambr), Tc (ambr)) for selection of supF plasmids. I can’t see supF on pCFJ350 so why would it be distributed in this strain?
Does growth on Amp alone result in loss of P3. If not, can P3 DNA get co-purified with pCFJ350 during mini-preps?
The bacterial strain designation at Addgene is a mistake - and I see that the mistake is propagated in several of the plasmids descriptions. We don’t even have the TOP10/P3 strain in the lab… The bacterial strain should have been TOP10 from Invitrogen without any additional plasmids in there except for pCFJ350. I’ll ask Addgene to correct it - thanks for pointing that out.
Cheers
Christian
Ok, I talked with Addgene. I submitted plasmid DNA to them and somehow those plasmids were transformed into Top10/P3 cells instead of Top10 cells. So, the bacterial stabs that have already been distributed contain the P plasmid in addition to the correct plasmid. Addgene are retransforming the plasmids and will distribute the plasmids in the correct bacteria.
For those that already have the plasmids this is not a big problem. Top10/P3 cells are Top10 cells that have been transformed with the P plasmid. The P plasmid is a 60 kb, low copy plasmid that contains KanR as well as AmpR(amber) and Tet(amber) resistances. AmpR and TetR are only functional when the cells are co-transformed with a plasmid that carries an amber suppressor (supF). None of the pCFJxxx plasmids contain this suppressor, so there should be no selection to keep the P plasmid in the cells. So, if you struck out the colonies on an Amp plate and grew up a culture under Amp selection then chances are that almost all of your DNA is from the correct plasmid.
If you want to get rid of the Top10/P10 background simply streak out the bacteria from Addgene through a couple of growth cycles on an Amp plate. Pick a single colony and grow up. To verify that the P plasmid is gone make sure the bacteria cannot grow on a Kan plate (the AmpR(amber) reverts at about 5% frequency so you could be selecting for a revertant).
See here for a link to the description of the cells from Invitrogen:
http://tools.invitrogen.com/content/sfs/manuals/oneshottop10p3_man.pdf
Sorry for the inconvenience
Christian