I've tried Gibson assembly master mix with NEbuilder positive control, and after transformation into DH5a cp cell, I spread them on the ampicillin medium and incubated at 37 Celsius degree for 16 hours and there were colonies.
However, when I extracted plasmid from them and linearlized plasmid with Ecor1 and performed electrophoresis, DNA size was around 2.7kb, which is just as pUC19 plasmid. So I performed electrophoresis them with pUC19 which was linearlized by Ecor1, both showed the same DNA size.
So next time I took DNA from NEBuilder Positve control solution directly (didn't even mix with Gibson assembly master mix and there was no reaction) and transformed into DH5a cp cell, and spread them on the Ampicillin medium. As far as I'm concerned, there should have been no colonies, but there were so many.
So I extraced plasmid from them and linearlized with Ecor1 and performed electrophoresis, and DNA size was around 2.7kb, just as pUC19. Is there any posibility that NEBuilder positive control solution might be pUC19?
*** I used NEB Gibson assembly master mix, and it says positive control should be 5.2kb
Thank you.
IMHO, you are obsessing and going down an uneccessary rabbit hole. I have probably personally designed and cloned 200+ unique constructs using a Gibson approach and i have never bothered with using the kits positive controls. As a generality, these Gibson kits are pretty full proof if your in-silico design is bang on. Have you tried a new kit's positive control and made sure that there wasn't a QC issue with that particular positive control and it was simply just an undigested/unlinearized pUC19. I'd suggest dropping these investigation and concentrate on your actual cloning ;)
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