So Nightmarish!

It took a week for me to figure out what happened to the T1T1-P cells supposedly harboring the pE2-MaSpCL. And finally, everything was laid crystal clear. After comparing the nanopore sequencing result (contig containing only the vector backbone sequence in tandem repeat except for the MSC site) with the plasmid profile from both single and double digestions, the insert was missing from the plasmid! This was also corroborated with the failure to amplify the CT band, false positive extended CT seq, NT seq as well as the repeat motif seq. Gosh, what has happened to my cells??? I did found a discussion on research gate that if the insert is somehow deleterious, the cell will remove it from itself but keeping only the plasmid. And also probably due to the instability of the plasmid to hold the insert..is the spidroin insert toxic to my cells? I do not know which one but definitely after repeated subculturing, my pE2-MaSpCL disappeared all of the sudden. I need to go back to the original stock culture hoping that the insert is still there..O mai, I am so worried sick now...The one and half year work!

                            I  was deceived by this profile!!! Nothing but a plasmid multimer! Arghgh..