Poor Maxiprep DNA yields of pSpCas9(BB)-2A-Puro (PX459)

[james.f...@gmail.com]

I've successfully cloned in my oligos into PX459 and confirmed this by sequencing.  Now when I try and scale up the clones using EndoFree maxi preps (i.e. Qiagen Cat#12362), I don't get any DNA, despite the fact that 2 maxi preps of other DNAs worked up on the same day had plenty of plasmid.  When I check the starter cultures I used to inoculate the maxi prep cultures, I see the DNA as purified using the Qiagen mini prep kits.  So something ain't right!  Anyone have this problem with pSpCas9(BB)-2A-Puro?  BTW, I've tried both Stbl3 and DH5a cells.

thanks very much

Jim Fessenden

[Phil Abbosh]

man that is a head scratcher.  i had a similar problem several years ago and called qiagen who said they changed something in their columns. they couldnt really offer advice at the time.  have you spoken to qiagen?  their tech support is (in my opinion) top-notch for the last year +.

also, Addgene does describe pX459 as "copy number: unknown."

were your other 2 same-day plasmid preps also pX459+insert?  pX459 without insert grew fine and gave plenty of yield?  if your problem-plasmid gives colonies on your streaked plate and your initial miniprep gave adequate yield (assuming you did that to sequence the construct), it wouldnt seem like a bug strain problem.  it seems like you have it narrowed down to the kit, or something about growing a larger prep with that particular plasmid. Because the kit works fine with your other plasmids, maybe the problem is culturing a larger volume?

i highly recommend the plasmid-plus midi kit (#12943) if you have access to a vacuum manifold and cant get the maxiprep kit to work.  35 mL overnight culture routinely gives 150-250 ug of plasmid in <1h.  do not use a conical tube for your overnighter though.

[james.f...@gmail.com]

Thanks for your detailed reply Phil.  I got the prep today, though the yield is quite low.  My guess is that it's a low copy # plasmid and I was getting faked out by the low yield.  It's enough to do my transfections though!

[Rahul Arya]

I used Clontech Maxi kit (Macherey-Nagel) and got yield of ~800ng/ul (500ul) from 250ml starting culture (DH5a). The kit requires old school way of elution in 15ml buffer followed by Isopropanol precipitation. I resuspended the pellet in 500ul volume. If you can bear this inconvenience, then it might worth be a try.

Rahul