Hi everyone, I am doing gibson assembly and I tried 4 times but its not working. I checked my primers and all but everything looks fine. The kit which I am using is one year old. So could it be that the kit is no more functional or it has lost its efficiency?
I can assure you my year old Gibson kit works just like the new one. I actually compared them directly, with the same reaction, and there was no difference in colony number or correct clone number. Maybe something special happened to your kit (freezer defrost cycles?).
With Gibson, not every reaction works, even if you do everything correctly. So the most likely explanation is that your new reaction simply cannot work due to sequence-dependent problems. That's one reason they included a positive control assembly with the kit. If that works, and still gives you the large number of colonies it used to on day one, then it's not the kit's fault.
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