Hi everyone!

I have been trying to do a cloning of a plasmid that should , in theory, have been straightforward. I took a PMT_CID_V5_6His_Hygro plasmid from my labs' plasmid stocks and cut the V5 tag out to replace it with a GFP tag instead using a NotI and AgeI RE digest then I used T4 Ligase in 1:7 vector: insert ratio. Then I did an analytical RE Digest using PvuII which confirmed the successful insertion of GFP instead of V5. Afterwards, I amplified a MED25 fragment from gDNA via PCR and introduced the NotI and KpnI RE sites into my fragment then did a RE Digest and subsequent ligation to replace CID by MED25. This proved to be very difficult. I tried many times es with Top10 CaCl2, RbCl and electrocompetent cells. Eventually, I managed to get colonies with my construct and 2 analytical digests to confirm the identity of my plasmid using XhoI and HindIII one time and PstI in another. And, both confirmed thatchers plasmid is indeed the right one pMT_MED25_GFP_Hygro. I even transfected cells with Cenpc tagged by HALO by my plasmid and detected a GFP signal. However, because cloning the MED25 into the plasmid was exceptionally difficult I sent the plamsidfor nanopore sequencing and the sequencing results showed the my MED25 is in but my GFP tag is not!! Surprisingly, the V5 tag sequence was the one present.

I don't understand what may have gone wrong exactly. I would,d very much appreciate some input.

P.s the HALO ligand I used was HALO549. I; also, will attach the plasmid maps here.

Many thanks in advance ☺️