We are frequently using plasmids with a certain backbone. Due to circumstances, some of these were maintained in Ecoli MC1061 and others in DH5a. Recently, we experienced problems with digests & sequencing with some of our preps. It seems as if these occur when plasmid prepped from MC1061 is transformed into DH5a. After miniprep from DH5a, they don't cut as expected, and give poor sequence reads. Similar digests and sequence reactions on the MC1061 prep work fine. Already after miniprep, they look different on gel (non-digested), but we expected that considering the genotype differences of the strains. If the constructs are never passed through MC1061 (e.g. after cloning into MC1061 isolated backbone, transformed to DH5a), these problems do not occur.
I would like to know if you have any idea why this would be?
I guess this must be methylation-related but hard to say exactly how - MC1061 has two mcr mutations which might be responsible:
http://openwetware.org/wiki/E._coli_genotypes#MC1061
Which enzymes are not cutting?
which restriction enzymes are you using for cloning? These species use specific methylation sites to prevent the sue of specific restriction enzymes.
per your question, the sequencing quality and efficient digestion results always depend on quality of plasmid extraction kit or the method used, plasmid size, kind of plasmid, medium culture and etc.
based on my experience, you have to look the user manual of the used vector; if the certain strain is recommended, it is better to use it. commonly, by transforming the vector to the none-recommended strain, the concentration of extracted plasmid shows significant decreasing. how ever, quality of miniprep plasmid extraction and its handling can effect the sequencing results.
about the digestion problems, i am agree with Nicholas A Dopkins....
In addition, in DH5a, by using larger plasmid backbone, you have to increase incubation time to extract more plasmid DNA.
GOOD LUCK!!!
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