DNA quality and MosSCI insertion frequency

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:

Number of F1s

MosSCI insertion frequency


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.

Posted in Improvements
3 comments on “DNA quality and MosSCI insertion frequency
  1. Christian says:

    When using the Invitrogen kit, did you also not use the included RNAse and do a EtOH precipitation afterwards? Just curious as this was recommended when using the Qiagen kit. Thanks!

    • Christian Frøkjær-Jensen says:

      I just followed the protocol on the Invitrogen kit. Since it is supposed to give DNA that is clean enough for cell transfection, I figured there was no reason to change anything. There’s an optional step to get rid of endotoxins which require an additional buffer – I also did not do that. CFJ

  2. 72hourfor10 says:

    At the time of injections performed to quantify the insertion frequency of the miniMos transposon we were not aware of the increased frequency resulting from higher DNA quality and these injections were therefore all done with the Qiagen miniprep kit.

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