I have a 4.8 kb insert that I attempted to ligate into pCDNA3.1 (~5.4 kb). Transformed ligated plasmid into DH5alpha E. coli. Insert is flanked by HindIII and ApaI cut sites. I digested the ligated plasmid with HindIII and ApaI and the insert popped out of my plasmid. On the gel I saw two bands: ~11 kb (presumed to be undigested plasmid) and a 4.8 kb band (presumed to be insert). I sent these positive clones for sequencing and the results indicated that my insert was not present.
Any explanations?
What is that insert? Did you express it? Did you double check the sequencing?
Hi Chandra,
We redid the sequencing and the same results came back. I cannot give too many details on the insert. The insert is not meant to be expressed in bacteria.
It may be obvious, but have you double-checked that these restriction sites are only present flanking your insert and not somewhere else in the backbone?
It is unlikely that you have non-clonal DNA because then sequencing would not work, so my next question is when you do analytical digest of many clones, do you see only positives (would indicate the band you see is not your insert) or also some negatives (no band)?
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