I fragmented the gene into 2 pieces but I could not PCR-amplify the fragments from (cDNA). I then fragmented it into 5 pieces and amplified the fragments but the gibson assembly did not work (just clones of empty vectors). To improve the efficiency, I merged some fragments to have 3 pieces of inserts, did 4 gibson assembly reaction (4 x 20 ul), run on agarose gel, cut out and extract the band corresponding to the desired construct, and used it to transform E. coli but I could not get a single colony.
Maybe try NEBuilder® HiFi DNA Assembly. It has several advantages.
In the process have you used Mg+2,?
Do you mean you cannot insert it onto the plasmid? Maybe by the enzymes, (blunt?) change the cassettes for antibiotics.
Mª Angeles Zorrilla Lopez-Perea Thank you for your suggestions. I currently use NEB 2X Gibson Assembly master mix. I will add Mg+2 to see if it works.
I could not amplify the full length (5.2 Kb) from cDNA, so I fragmented the gene.
The digested vector has overhanging sequence at both ends.
Mª Angeles Zorrilla Lopez-Perea I have been trying to clone 2 PCR fragments into a 5Kb vector with NEBuilder® HiFi DNA Assembly. But so far, not working.
What do you mean by using Mg2+? For better polymerase activity?
Mg+2 stimulates any process related to cloning. There might be more,but it is normally Mg+2. It could mean either not working or working properly.
So just adding Mg+2 to the assembly mix might stimulate the process? Even with the kit?
What about the concentration of Mg+2? Is there any already established one?
Depending on the processes, there is a fixed amount of Mg+2,but it is important to have them in all
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