I've also been doing some SLiCE experiments with SLiCE made from DH5alpha E. coli with and without pKD46 plasmid that I got from the-odin.com. SLiCE was used to successfully ligate two pieces of DNA together. The efficiency was fairly poor when using SLiCE from E. coli with pKD46 (12 colonies from 108ng of DNA, where the commercial kit NEBuilder produced ~500 colonies). The SLiCE from E. coli without pKD46 produced many more tranformants, but some/many of these seem to be original vector that was able to religate after being cut (vector was neither gel purified nor phosphatase treated, which I have found to be unnecessary when doing Gibson/NEBuilder assemblies). Consistent with this, no SLiCE controls also contained many colonies that sequenced as the original re-ligated plasmid. Spontaneous re-ligation seems to occur frequently, yet the lambda exonuclease in pKD46 (and in NEBuilder) seems to prevent religation of digested vector, yet it is not clear from these results that pKD46 aides in efficiency of ligation.
On Tuesday, November 17, 2015 at 6:15:49 PM UTC-6, Koeng wrote:
-- Full experimental details and results/pictures are in this Google Doc.
On Tuesday, November 17, 2015 at 6:15:49 PM UTC-6, Koeng wrote:
(meaning I got ~10x more colonies with 20bp vs 40bp)
On Tuesday, November 17, 2015 at 11:12:27 AM UTC-8, Nathan McCorkle wrote:On Mon, Nov 16, 2015 at 7:06 AM, Koeng <koen...@gmail.com> wrote:
> Recently I've begun using SLiCE for cloning and have got some of my labmates
> to jump aboard. From about 1-2 weeks of trials, here's what I've found with
> single part SLiCE assembly. In the future, I hope to try 1. PEG8000 to
> increase SLiCE efficiency and 2. Multipart assembly 3. using homemade comp
> cells.
>
> Important stuff: SLiCE is approx. 1/3 the efficiency of homemade gibson
> assembly. Gibson increases efficiency with additional homology while SLiCE
> decreases. The optimal homology length for gibson is ~40 while the optimal
> homology length for SLiCE is ~20. SLiCE
Does you last sentence mean SLiCE won't work with more homology? Have
you verified this?
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