pCW cloning by RE ligation with weird product

We are trying to subclone a ~4kb PCR fragment into the pCW-puro/blast backbone (~6kb). Both fragments run at expected MW have been gel-purified. The ligation reaction appeared to have worked efficiently, as the backbone only plate had 0-5 colonies, while the insert-containing ligation yield hundreds of colonies. However, when we picked 30 or so colonies from the Amp plate for miniprep, all the resulting plasmids DNA looked similarly weird: 1) there seem to have a mixture of DNA products; 2) the dominant species runs at 1.4kb or 2.1 kb.

We appreciate it if you can share with us your experience and suggestion!


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Red Dragon3 months ago

Maybe you can ____

  1. Cut by some restriction enzyme then run gel __ __
  2. Send to plasmidsaurus for NGS. __ __

You can further troubleshooting once you know what you get. __ __



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