I've been doing some RF/Gibson cloning, and I am having issues sequencing my plasmids. I performed colony PCR to identify clones with the appropriate inserts. I then grew these up and plasmid prepped them to extract the DNA, which I then quantified using a Nanodrop. When I sent these plasmids for sequencing, the reactions come out as complete trash (ie. a bunch of "unknown" base pairs, or just failed sequences in general). I tried a few troubleshooting techniques:

1) Changing the amount of DNA I prepare for sequencing (according to the guidelines set out by the sequencing company).

2) Using the sequencing company's standard primers instead of adding in my own.

3) Re-streaking the frozen stock and ensuring that I select only a single colony, which I then grow up for plasmid prep.

For all of these, the results still came back the same (ie. bad). I then PCR'd a region that covers the insert, Dpn1 digested it, cleaned it up, quantified via Nanodrop and sent that for sequencing. This time, I got perfect results (ie. everything was exactly as desired).

I am wondering what changed? Why are the plasmid sequencing reactions failing, but a PCR of the plasmid isn't? I have to create 12 clones and it will be quite tedious to PCR all my clones prior to sequencing, rather than sequencing them from the plasmid directly.

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