Increase in Cloning Mutations

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Increase in Cloning Mutations

Postby marchelko » Apr 27 2017 11:21 pm

Hello,

I've had a dramatic uptick in the frequency of mutations in my plasmids recently (couple of months). This has included point mutations, small ~100nt recombinations, and larger multiple kb rearrangements. I am used to seeing mutations maybe once or twice a year (if that) but now almost every plasmid has some mutation. These mutations occur with Gibson Assembly, GoldenGate, and old fashioned hard cloning. I'm also using NEB Stable E.coli which have worked usually worked great.

I've replaced my water and made new reagent mixes but that has not solved the problem.

Has anyone had similar issues and figured out what was going on?

Thanks,
-M
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Re: Increase in Cloning Mutations

Postby r.rosati » Apr 28 2017 4:06 pm

This could be explained if you're making your own competent cells and your last batch was contaminated by other bacteria (after all, NEB Stable have no antibiotic resistance). If the rogue bacteria incorporate the plasmid and delete a part of it, they'd get a growth advantage towards Stable cells (they might even have an advantage upfront because they're not a "tamed" strain).
Could this be a possibility?
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Re: Increase in Cloning Mutations

Postby marchelko » Apr 28 2017 5:22 pm

Thanks R. rosati,

I had my suspicions that this may be the case but had not followed up since other lab members have not had similar challenges. I'll give a fresh stock of competent cells a shot anyway since my constructs contain more short repeats which have a greater likelyhood of generating recombination.

-M
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