Has anyone got recommendations for miniprep kits to purify plasmids for microinjections?
Our lab currently uses the “Invitrogen™ PureLink™ HQ Mini Plasmid DNA Purification Kit” with success but the expected delivery time of a new kit keeps changing so leaning about a good alternative might be useful.
Although we recently published the below paper addressing this very question in worms, we too have had a problem with the Invitrogen kit being backordered since April. So we switched to the standard Qiagen miniprep kit (with the PB wash included) and have so far been able to reproduce the results described for the Invitrogen kit + PB wash in our paper. We continue to get multiple knock-ins from injecting just 10 worms. This was a pleasant surprise because the Qiagen kit costs us just $1.29 per prep vs. the $5.07 per prep for Invitrogen PureHQ. Hope this helps!
I use Qiagen miniprep DNA for all the transgenes I create using RMCE. In addition, I re-use the columns ( after an acid wash, Tris neutralization, and TE wash- multiple publications describing this approach) and use DIY reagents which leads to very affordable column DNA preps. I get approximately 5-6 sqt-1(e1350) rol animals per injected animal (one gonad arm, n> 200 injection session of 12 animals) with Qiagen DNA. The tritonX-114 paper would suggest that one can do better, but that paper does not assess whether the increase in F1 transgenes actually yields an increase in lines or integrants. And I personally see little correlation between the number of F1 Rol animals and the freq of F2 integrated lines in my experiments. This is probably because some of the construct I inject are transcriptionally toxic (just like a pCFJ90- which in my hands become exceedingly toxic at above around 2ng/ul in an injection mix).
Thank you all for your suggestions. We will try the standard Qiagen QIAprep Spin Miniprep kit with the “omit RNaseA and do an ethanol precipitation” approach, once we have run out of our final few Invitrogen columns. I will update this thread with our results once we get to this.
We use Qiaprep mini spin or midi kits without issues, I imagine with similar results to the invitrogen kit. I have also used monarch and zymo kits, honestly I haven’t had a miniprep kit which doesn’t work for injection.
We’ve also felt the miniprep supply chain delays. We are big fans of the Biorad Quantum miniprep kit, however we’ve been waiting months for it to arrive. We currently use Qiagen Mini kit and have been able to generate knockins. However, we found that we cannot use the Promega Wizard kit to prep DNA for injections.
There are decades of complaints about the Promega Wizard kit. I remember the official user’s manual for an ABI sequencer saying it wouldn’t give DNA that could be sequenced - and that must be 25 years ago now.