Re: [DIYbio] Re: Gibson vs SLiCE (single part recommendations)

Bryan, thank you very much for sharing your experiences. Sounds like you did all the right controls to get a proper assessment of the system. Logically it is a little surprising that there's no carry over of plasmid, but of course very good news and just goes to show the best way to answer a question is to just do the experiment. I tend to see too many people over thinking things to a point where they will convince themselves something is not feasible before they've even tried it, so the project stops before it even begins. :)

Cheers.

On Monday, March 13, 2017 at 11:57:49 AM UTC-7, Bryan Jones wrote:
I would imagine there is some risk of that, but I haven't seen that happen in my experiments. I carried out a negative control using just SLiCE extract with competent cells (No insert DNA), and didn't get any colonies on amp plates. Also the colonies I sequenced from my tests were all correct, no pKD46 carryover. It might be that DNAse breaks all the DNA from the SLiCE cells. Normally when you do a plasmid prep, you add EDTA to prevent this, but no EDTA was added here. I'm not positive why, but empirically, I can say that it does not seem to be an issue. 

On Tue, Mar 7, 2017 at 6:16 PM RBC <rco...@gmail.com> wrote:
Is there any worry that the amp resistant pKD46 plasmid will be extracted into the SLiCE lysate, in which case this plasmid will carry through to the final transformation? To me it would seem you can't use it to make amp resistant vectors..or maybe I'm missing something. Thanks for the clarification.

RC


On Friday, October 21, 2016 at 11:22:11 AM UTC-7, Bryan Jones wrote:
In my most recent test, without pKD46, I got 10 colonies from the SLiCE reaction, which was the same as the no SLiCE control. SLiCE with pKD46 resulted in 50-60 colonies. For comparison, NEBuilder resulted in about 100.



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