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
Hi Rakesh, thank you for your reply. DpnI digestion was performed not to digest PCR product but to destroy the carry over plasmid template DNA used in PCR reaction.
okay, I would like to suggest then think on concentration and quality of enzyme and template amount.
Dear Mahmudul,
Give it up. Buy the GeneArt Gene Assembly System from ThermoFisher Scientific and try it out. I routinely assemble 7 parts in one reaction.
Best regards,
Wes
Simple question, but did you design all of your gblocks with the appropriate overlapping overhangs?
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