Re: [DIYbio] Re: Trible digest - ligation

What I did so far...

I chose just to insert the insert into one vector, and screen the transformatns for GFP activity. Later I will add the other vector with another restriction site, and then plate transformants on agar with both antibiotics... 

Did one ligation for ~ 45 minutes at 37°C and one at 4°C overnight... 

On Mon, Apr 22, 2013 at 2:46 PM, poli <polinere@gmail.com> wrote:

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:

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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