I've been cloning some inserts into a lentiviral vector (pBOB backbone) by RE digestion and blunting ends with T4 DNA polymerase.
After these steps, I usually load the digested product onto 0.7% agarose gel to isolate the expedcted size fragments. Unexpectedly, during agarose gel separation the bands from the lentiviral vector are much less intense compared to the insert ones.
Having exctracted the DNA from such a band, its amount is not sufficient to pursue the ligation process (I'm getting around 0.5 ng/ul of vector!).
I have already repeated the digestion three times and always get the same profile. I've also been checking the DNA amount after each cloning step (e.g. RE digestion, fill-in) and I notice that the lentiviral vector DNA amount is always half the insert DNA, which is always ok and in a sufficient amount to be ligated.
Having used the same reagents for either vector and insert DNA, I would actually exclude a technical issue, as I'm taking the impression that there's something wrong with the lentiviral DNA. Of note, it is a purchased plasmid from Addgene which I have already checked by RE digestion.
Do you have any idea of what's going on? Also, do you have any suggestions about how to fix the problem? Thank you in advance for your kind help!