Cloning issues with viral vectors

Virology Viral Vectors
1

Hi I am trying to insert my gene of interest (which is rather large…~4kb) into a viral vector. I first started with pBABE-puro viral vector (~5.1kb) but had no luck. I tried inserting a different, much smaller gene, and it went in without a problem on the first try. I figured the size of my gene was the problem so I moved to the slightly larger pLXRN retroviral vector. Again, after many tries, I could not get insertion of my gene. I have tried over and over. I’ve tried new restriction enzymes, different vector DNA ratios, different ligation temperatures, etc. I always dephosphorylate my vector with antarctic phosphatase as well. I usually end up with more colonies on the vector+insert plate, but when I screen them, there is no insert.

Does anyone have any suggestions?

2

Daniel Suarez

You can try the following:

Optimize Ligation Conditions

  • Insert:Vector Ratio: Use a higher molar ratio of insert to vector (e.g., 3:1 or 5:1) to increase the likelihood of successful ligation.

Vector and Insert Size Considerations

  • Large Inserts: Cloning large inserts (>4 kb) can be tricky due to lower ligation efficiency and potential instability in bacterial hosts. Viral vectors like pBABE-puro and pLXRN may struggle with larger inserts due to size constraints or secondary structures.
  • Vector Choice: Consider using a vector specifically designed for large inserts, such as:
    • pLenti or pLKO (lentiviral vectors, which can accommodate larger inserts).
    • BACs (Bacterial Artificial Chromosomes) if you need even larger capacity.
5 Likes