I have had a lot of success with Gibson reactions in the past, but all of a sudden, not only has my number of total colonies fallen, the percent of correct colonies has also fallen. I am trying to assemble three fragments (900bp, 780bp, 300bp) and a backbone (9.5kb), and I do still get colonies. Most of them look good by restriction digest, but the sequencing results now always show random point mutations that differ from colony to colony, with some having only one mutation, others 5+. I got all of my fragments sequenced and I know that before the assembly, they have really clean reads. It seems that these mutations are happening either during the assembly or the transformation. The mutations are often, but not always, near a "seam" where two fragments meet. I thought at first the polymerase in the master mix had gone bad, but I have tried buying fresh NEB Gibson Master Mix, and the same kind of mutations still happen. (Side note: my last two orders of master mix from NEB have smelled bad- almost sulfuric? Did I just not notice this before or is this a sign the mix is tainted?) The positive control still works and gives many colonies. Could it be my competent cells introducing the mutations?? Any help would be greatly appreciated!
It could be that your final construct is toxic. Therefore there is a selection for constructs mutated during the assemblies. The majority of mutations are around the seams since it is the most likely site to be mutated during this kind of assembly. If you want to make sure it is not your mix that is the problem try PCR amplifying the full fragment from the GA mix and sequence that. If you find that it is clean it is then probably a toxic construct.
maybe you could try hifi?
NEBuilder HiFi DNA Assembly offers several advantages over NEB Gibson Assembly. These include: higher accuracy due to the use of a high-fidelity polymerase, the ability to assemble both 5´- and 3´-end mismatches, lower DNA input requirements and the ability to bridge two dsDNA fragments with a ssDNA oligo (data not shown). NEBuilder HiFi is the clear choice for efficient and accurate DNA assembly.
It should smell rotten. That's an SH-compound like bME or DTT to keep the enzymes in a reduced state. This smell would get lost during long term storage as the compound oxidizes or evaporates. It's totally expectable that new mix would have more of it than old mix. That's all very accurately observed, and a good thing.
I've never seen mutations like that. Have you sequenced the same clone twice and both ways, to rule out sequencing errors?
John Schloendorn Phew! That's good to know! I thought I was going crazy.
I have at least two reads for each mutation, and the sequencing always shows the same mutations within the same clone. The peaks are also very clean, so I am pretty convinced they are not a product of the sequencing.
Hanna Alalam That is a great idea to PCR from the GA. I will run a reaction that spans at least two fragments to see if the product is already mutated.
Junjie Shao Wow how have I never heard of HiFi?? I just looked it up, and it definitely looks worth trying to reduce mutations if it does turn out that the mutations are happening during assembly.
I have the same problem, mutations happen at increased frequency at the Gibson Assembly junction, but I don't know why. You meanwhile found out if HiFi working better for you? If others don't think this is normal, I must assume its my personal GA protocol or 42 degree heat shock problem. Or eventually the quality of the primer?
- Badges
- Science topic