Hello. Recently I've been assembling plasmids that use pSMPUW as the backbone (kanamycin resistant). I've noticed that the minipreps for these plasmids usually sequence fine (I've been using stbl3 cells), but once I midiprep the clones, I get poor sequencing results for these samples (smears, no priming). Has anyone had this experience before?
Have you nanodropped your midipreps to check their quality and concentration?
If you have done that already, and nothing is obviously wrong I'd try digesting some of your midipreps with a restriction enzyme (or two) and check if you have expected band sizes as well as loading a well with just the undigested plasmid.
If you get bands with restriction digest but they aren't the expected size then you just have incorrect clones. If you get a smear/nothing in the restriction enzyme digest but have visible bands for undigested plasmid then you probably have endonuclease A contamination, maybe the midiprep method you're using doesn't do a good job of removing it.
If you get smears/empty lanes in both then you probably have contamination with either genomic DNA, low molecular weight nucleic acids or RNA which means something is probably going wrong at the lysis/neutralization steps, or the RNAse A has gone bad.
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