Dear Haja,

There is no real good explanation for your problem. There is only this unusual BstXI enzyme which will work only in case your insert and your vector have an identical overlap of 4 nucleotides. If this is the case it may be possible tat someone finds a clue when you add your gel pictures.

Best, Uwe

you might be creating an unstable construct in E.coli I would suggest you try a strain for unstable plasmids like stbl3 and do the recovery and growth steps at 30C.

Are you certain that the digested out insert product is ~1500 bp and your vector is still 9508? It's challenging to resolve both a 1000-1500 bp product and 9508 product accurately on the same gel. I suspect that either your insert is longer than you think OR that there is another cut site in your vector that is adding length to the insert.

Do your BstI1 or NotI cut sites directly flank your insert or are they ~500 bp away?

Have you tried sequencing your insert? Amplify the insert using primers that anneal to the vector surrounding the insert and sequence from both directions. That will tell you the size and exact sequence of your insert.

Good luck!

You could try different enzymes or a PCR to confirm. You could have an incorrect plasmid map. What happens if you digest the insert negative vector with the same enzymes?

It's astounding to me that in 2019, people are still stubbornly approaching cloning in this way. Diagnostic digests are absolute dinosaurs in this realm. Colony PCR or just some cheap 3rd party sanger sequencing are all available to you to complete resolve your issue.