Competency of cells shouldn't matter much, the ligation reaction needs to be efficient. Ligation reactions are inefficient due the small section of complementarity. To increase the efficiency use high concentrations of ultra pure DNA- gel filtration with extra wash step, use rapid ligation buffer, incubate overnight, and possibly add a second round of atp and enzyme.
Alternatively you could fusion PCR or gibson the three pieces together efficiently.
On Monday, April 22, 2013 3:17:51 AM UTC-4, Mega wrote:
-- On Monday, April 22, 2013 3:17:51 AM UTC-4, Mega wrote:
Hi,I have three plasmids and want to combine them into one...May this work in one step?two of them carry two different antibiotic resistances, the other is a GFP cassete flanked on both sides. So selection shoud be quite easy...I would just cut those three plasmids (two of them will just be opened, one cut two times) without having them on a gel, and then ligate them together... The clone that grows on both antibiotic and fluoresces will be the one.Is it vital to use ultra-competent E.Coli?
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