Morris Maduro has previously pointed out that the quality of the DNA preparation for injection made a large difference in the number of rescued F1 progeny (see Worm Breeders Gazette). Since we had started seeing some variability in the insertion frequency, I tested different mini and midiprep kits for toxicity and insertion frequency.
I tested three kits:
- Qiaprep Spin miniprep kit (cat. no. 27106) (with RNAse and no EtOh precipitation)
- Qiagen Plasmid Plus Midi kit (cat. no. 12943)
- Invitrogen PureLink HQ Mini Plasmid Purification Kit (cat. no. K2100-01) (Mihail Sarov had pointed out that this kit is very efficient in his hands)
Experiment: I injected a 4.6 kb transgene (Peft-3:GFP:tbb-2 cb-unc-119) for insertion at the ttTi5605 site. I prepared all the DNA for the injection mix with each kit including the co-injection markers. (pCFJ601, pMA122, pGH8, pCFJ90, pCFJ104). I did three sets of injections of 20 animals for each construct.
Here are the results:
Data shown is mean +- SEM.
As you can see, the Invitrogen kit performed best in terms of F1 animals and insertion frequency. The midikit also did well, whereas the miniprep kit from Qiagen clearly gave reduced number of F1 progeny and lower insertion frequency.
So, although the Invitrogen is a little spendy, it may be worth it for making plasmids for injection.