Hi All,

I am trying to generate a bacterial operon construct. I have designed four gBlocks (each of which is ~1.7 kb) and synthesized them from IDT. I am using pGex-6p-1 vector to assemble these gBlocks using Gibson assembly. I prepared my vector by PCR (primer designed by NEB builder software). After PCR amplification, I checked 5 ul of my PCR product in the gel and found a nice clean single band and the size of this band matches to the expected size (for details see the attached PPT slide). After that, I purified the PCR product using Promega PCR and gel purification kit. I eluted in 30 ul of nulcease free H2O and then measured the concentration of PCR product using NanoDrop and the concentration was 296 ng/ul. Then, I digested 8 ul (296 ng/ul) of purified PCR product with DpnI (1 ul) in a 10 ul of total reaction volume (and incubated at 37oC for 30 mins) as suggested by NEB Gibson manual and heat inactivate the DpnI at 80oC for 20 mins. After that, I set up a Gibson assembly following the instruction of NEB Gibson manual. I incubated the Gibson reaction at 50oC for 1 hr in a PCR machine and then transformed 2 ul of assembly reaction in 50 ul of NEB 10-beta cell (High efficiency) following the transformation protocol for NEB beta cell as specified by NEB. In the following day of transformation, I found many colonies (see attached PPT for picture). I took five well separated colonies and then grew a 5 ml overnight culture. After that, I isolated plasmid DNA using Promega plasmid purification kit and digested them with SalI-HF enzyme which should give three bands if there is a assembled product. Otherwise, they should produce linear vector as pGEX-6p-1 has an internal SalI site. I then checked 60 colonies by colony PCR and it seemed that all of the colonies I checked by colony PCR contains the intact pGEX-6p-1 ( I did not include this gel image in the PPT slide). What things might be wrong with my cloning? Specifically, I would like now

i) Why I am getting intact pGex-6p-1 whereas I generated the linearized the pGex-6p-1 by PCR and digested the PCR product with DpnI? Is digestion not working??

ii) What might be the good way to assemble these four gBlocks into pGex-6p-1 vector?

iii) How can I reduce the vector only background?

iv) I would also highly appreciate any suggestion on my strategy that I am using to generate this operon construct.

For details description of my cloning strategy, please see the attached PPT slides.

Thank you all,

Hassan

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