I am doing some cloning and am having some issues with both sequencing my 22kb plasmid and running digestions with it.
I sent my plasmid in for sequencing and got terrible results, but submitting it to Macrogen's "difficult sequencing" protocol would up working great.
It turns out that my plasmid has repetitive sequences, and GC repeat rich regions, and likely other issues (secondary structure) that were keeping it from sequencing correctly.
I am also having issues with digestions for this plasmid, likely due to the same issues. Does anyone have any suggestions for other reagents I could add or different procedures that work well for digesting recalcitrant plasmids?
Thanks!
I'd suggest setting up a larger volume digest (50-100 microliters), adding BSA to your digests, letting them incubate overnight, and trying a variety of endonuclease enzymes to find one that works well. Basically, try the "old school" method of setting up a digest.
Good luck!
If you have repetitive fragments, there maybe multiple enzyme sites. Try different enzymes.
I have faced the same problem. Afterwards i told company people to add 10% DMSO in the reaction mixtures: they were setting up for amplification of the gene i gave them for sequencing. It worked very well for me. My genes of interest were from M. tuberculosis which are high in GC content. If possible, you can also recommend them to add 10% DMSO in the reactions you are setting up for sequencing experiments. All the best.
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