Hi everyone:
I am trying to ligate my GOI with adeno-xl Tet-on vector. It seems to be quite a tricky kit. I have constructed my recombinant shuttle-GOI plasmid and checked it with many different combinations of restriction digestions as well as PCR. All gave me desired results. However, when I cut this recombinant plasmid with I-Ceu and PI-Sce I, I got two bands 4.8kb and 3.4kb. However, I only expected the band sizes to be 2.8kb (the off-cut of the plasmid, should be the same size for all of us, right?) and 3.8kb (my GOI in the cassette). Surprisingly, if you add 4.8 and 3.4 is even bigger than 2.8+3.8. Isn't this ridiculous? Where could have this big plasmid ever been coming from? When I thought about this, I realized that I did not run the digestion product immediately, as my LD did not have SDS added. The two bands (4.8kb and 3.4kb) were given from the purified digestion product using the phenol-chloroform method same as manufacture's protocol. I then ligated the GOI fragment with adeno-x at 16C for 16hrs, cut with SwaI for 2hrs at 25C and transformed using DH5a, but on the next morning I observed nothing on the plate.
Do you have any idea of what might have happened according to your experience? Many thanks
Jarod
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